Recombinant viruses displaying a nonviral polypeptide on their external surface

ABSTRACT

We have made retrovirus particles displaying a functional antibody fragment. We fused the gene encoding an antibody fragment directed against a hapten with that encoding the viral envelope protein (Pr80env) of the ecotropic Moloney murine leukemia virus. The fusion gene was co-expressed in ecotropic retroviral packaging cells with a retroviral plasmid carrying the neomycin phosphotransferase gene (neo), and retroviral particles with specific hapten biding activities were recovered. Furthermore the hapten-binding particles were able to transfer the neo gene and the antibody-envelope fusion gene to mouse fibroblasts. In principle, the display of antibody fragments on the surface of recombinant retroviral particles could be used to target virus to cells for gene delivery, or to retain the virus in target tissues, or for the construction of libraries of viral display packages.

FIELD OF INVENTION

This invention relates to recombinant viruses, also referred to asrecombinant viral particles. By "recombinant virus", we mean a virus inwhich at least one of the components of the virion particle is alteredor derived by recombinant DNA technology.

This invention also relates to the field of therapeutic gene transferand concerns the teleological design and use of vectors to deriverecombinant proteins or protein components suitable for display on thesurface of a gene delivery vehicle which, when displayed on the surfaceof the gene delivery vehicle, through their interaction with componentsof the surface of a eukaryotic target cell, are capable of influencingthe efficiency with which the gene delivery vehicle delivers its nucleicacid into the target cell, or of transmitting a signal to the targetcell which influences the subsequent fate of the delivered nucleic acid,and which are thereby capable of enhancing the suitability of the, genedelivery vehicle for an intended application.

BACKGROUND TO THE INVENTION

Display of a functional nonviral polypeptide on a virus which can infecteukaryotic cells

Recombinant viruses have been widely used as vectors for the delivery offoreign genes into eukaryotic cells. Recombinant viruses which are usedfor delivery of foreign genes to animal cells include members of severalvirus families, including Adenovirus, Herpesvirus, Togavirus, andRetrovirus families. Viruses which infect eukaryotic cells comprise aprotein shell or shells (the capsid) formed by the multimeric assemblyof multiple copies of one or more virus-encoded proteins. The capsidhouses the viral nucleic acid (RNA or DNA) and may or may not beenveloped in a lipid bilayer which is studded with virus-encodedoligomeric spike glycoproteins, visible on electron micrographs asspikes projecting from the surface of the virus.

The initial event in the virus life cycle is binding to the surface ofthe eukaryotic target cell. Binding is mediated by the directinteraction of specialised proteins or glycoproteins on the surface ofthe virus (antireceptors) with receptors on the surface of the targetcell, or indirectly via soluble ligands which bind the virus toreceptors on the surface of the target cell. In some instances, theinteraction between a virus and a target cell receptor may transmit ametabolic signal to the interior of the target cell. Binding is followedby penetration of the target cell membrane and entry of the viralnucleic acid into the cytosol (reviewed in Marsh and Helenius 1989 AdvVirus Res 36 p 107-151). Some nonenveloped viruses undergoconformational changes which result in their direct translocation acrossthe target cell membrane, whereas others, such as adenovirus, are firstendocytosed and then cause disruption of the wall of the acidifiedendosomal vesicle. Enveloped viruses fuse with the target cell plasmamembrane whereupon the virus capsid (or core particle), housing theviral nucleic acid is released into the cytoplasm of the target cell.This envelope fusion event is catalysed by oligomeric viral membranespike glycoproteins which are anchored in the viral envelope and may, ormay not be dependent on the prior endocytosis of bound virus and itsexposure to an acidic environment within the endosomal vesicle. Themechanisms by which viral spike glycoproteins catalyse membrane fusionmay involve their proteolytic cleavage, the dissociation ofnoncovalently linked subunits or other conformational alterations whichexpose buried hydrophobic moieties capable of penetrating the lipidmembrane of the target cell. Thus, virus-mediated delivery of nucleicacid is a complex, multistage process.

After delivery of the viral nucleic acid into the target cell, furthersteps in the viral life cycle which lead to viral gene expression,genome replication and the production of progeny viruses are oftencritically dependent on variable host cell factors. For example,division of the infected target cell is required for efficientintegration of a reverse-transcribed retroviral genome into the hostcell chromosome and subsequent retroviral gene expression (Harel et al,1981 Virology 110 p202-207).

The spike glycoproteins of one virus can be incorporated into the viralparticles of another strain. Thus with dual viral infection of a singlecell by two enveloped mammalian viruses, the host range of either virusmay be predictably extended due to promiscuous incorporation of spikeglycoproteins encoded by both viruses. This has been shown for closelyor distantly related retroviruses (Levy, 1977 Virology 77 p811-825;Weiss and Wong, 1977 Virology 76 p826-834; Besmer and Baltimore, 1977 JVirol 21 p965-973; Canivet et al, 1990 Virology 178 p543-541;Lusso etal, 1990 Science 247 p848-852; Spector et al, 1990 J. Virol 64p2298-2308) and for unrelated viruses from different families (Schnitzeret al, 1977 J Virol 23 p449-454; Metsikko and Garoff, 1989 J Virol 63p5111-5118; Schubert et al, 1992 J Virol 66 p1579-1589). The spikeglycoproteins of certain closely or distantly related retroviruses havealso proved to be entirely interchangable, allowing production ofinfectious hybrid virions with envelope spike glycoproteins of oneretrovirus and core particles of another retrovirus (Mann et al, 1983Cell 33 p153-159; Wilson et al, 1989 , J Virol 63 p2374-2378). Similarresults have been demonstrated with insect/plant viruses (Briddonp2374-2378). Similar results have been demonstrated with insect/plantviruses (Briddon et al, 1990 Virology 177 p85-94). Furthermore virushost range was predictably altered by exchanging the N-terminal receptorbinding domains of envelope spike glycoproteins from relatedretroviruses with distinct cellular tropisms (Battini et al, J Virol1992: 66 p1468-1475). When viral spike glycoproteins from closelyrelated virus strans were coexpressed in the same cell, mixed oligomerswere formed with high efficiency (Boulay et al, 1988 J Cell Biol 106p629-639; Doms et al, 1990 J Virol 64 p3537-3540). Thus, there isconsiderable scope for altering the host ranges of recombinant virusesby exchanging, mixing and recombining the viral spike glycoproteins ofnaturally occurring viruses.

However, a relatively small minority of the universe of eukaryotic cellsurface structures are actually used as receptors by naturally occurringviruses and it is often difficult to identify a virus with a host rangewhich coincides with the requirements of a particular gene therapyapplication. For example, to genetically modify a population of normallyquiescent haemopoietic stem cells in vivo, a most desirable genetransfer vehicle would be a recombinant retrovirus whose spikeglycoproteins do not bind to nontarget cells, but which bind tohaemopoietic stem cells and mediate membrane fusion and which alsosignal the target cell to divide as the nucleic acid is delivered. Thereare no known naturally occurring viral spike glycoproteins which meetthese requirements, and there is therefore a need for new technologiesto facilitate the generation of novel spike glycoproteins which canenhance the specificity and efficiency of virus-mediated gone deliveryand expression.

Preformed viral particles can be attached to cells which lack virusreceptors by way of a (multivalent) molecular bridge. This is clearlydemonstrated by the phenomenon of antibody-dependent enhancement ofviral infectivity. Thus, antibody-complexed foot-and-mouth disease virus(a nonenveloped picornavirus) has been shown to infect normallyinsusceptible cells via the Fc receptor (Mason et al, 1993 Virology 192p568-577). The phenomenon of antibody-dependent enhancement of viralinfectivity, mediated through binding of antibody-complexed viruses tocellular Fc receptors and complement receptors has been demonstrated forseveral enveloped and nonenveloped viruses (Porterfield, 1986 Adv VirusRes 31 p335-355). Moreover, bivalent antibodies that bind dengue virusto cell surface components other than the Fc receptor were recentlyshown to enhance infection (Mady et al, 1991 J Immunol 147 p3139-3144).Also, Baird et al (1990 Nature 348 p344-346) showed that herpes simplexvirion penetration into vascular cells via the basic fibroblast growthfactor (FGF) receptor requires the association of soluble FGF with theviral particles.

Goud et al, (1988 Virology 163 p251-254) incubated ecotropic murineleukaemia viruses (MLVs) with monoclonal antibodies against the gp70SUviral envelope spike glycoprotein and incubated human HEp2 cells withmonoclonal antibodies against the transferrin receptor. Crosslinking ofthe bound monoclonal antibodies with a sheep anti-mouse kappa lightchain antibody allowed the binding of virus on HEp2 cells and itssubsequent internalisation into the cells at 37° C. However,internalisation of the virus by this route was not followed byestablishment of the proviral state. Subsequently, Roux et al (1989 ProcNail Acad Sci USA 86 p9079-9083) and Etienne-Julan et al (1992 J GenVirol 73 p3251-3255)) used a similar approach in which biotinylatedantibodies against the murine ecotropic retroviral envelope spikeglycoprotein and against specific membrane markers expressed on humancells were bridged by streptavidin and used to link the virus to thehuman host cell. The method was successfully used to infect human cellswith ecotropic murine retroviruses bound to MHC class I and class IIantigens, and to the receptors for epidermal growth factor and insulin.However, targeting of the transferrin high density lipoprotein andgalactose receptors, and of various membrane glycoconjugates, by murineecotropic retroviruses did not lead to the establishment of a proviralstate.

Preformed viral particles can also be chemically modified to facilitatetheir binding to target cells which lack receptors for the unmodifiedvirus (Neda et al, 1991 J Biol Chem 266 p14143-14149). Murine ecotropicretroviral particles which had been chemically modified with lactosewere shown to bind specifically to the asialoglycoprotein receptor onhuman HepG2 cells (which lack receptors for murine ecotropic viruses).Binding was followed by retroviral infection of the human cells asindicated by transfer of a functional β-galactosidase gone.

Thus, the display of a functional non-viral polypeptide at the surfaceof the virus can lead to the preferential binding of the modified viralparticles to selected target cells, and in some cases, dependent on theexact specificity of the displayed polypeptide, binding is followed bydelivery and expression of the encapsidated viral nucleic acid. However,the present inventors realised that a method for the production of viralparticles which incorporate and display a nonviral polypeptide duringtheir assembly would be more useful, avoiding the need for modificationof preformed virions. Such a method would also facilitate the use ofsuch recombinant viruses as genetic display packages (see below).

Non-viral proteins have been incorporated, during assembly, into viralparticles capable of infecting eukaryotic cells. Thus, spontaneousincorporation of non-virus-encoded mammalian cellular proteins has beenobserved in retroviral particles. For example, MHC antigens areincorporated into the envelopes of human and simian immunodeficiencyviruses (Gelderblom et al, 1987 Z Naturforsch 42 p1328-1334; Schols etal, 1992 Virology 189 p374-376). Also, mammalian CD4 expressed in arian(quail) cells was incorporated into the envelopes of budding avianretroviruses (Young et al, 1990 Science 250 p1421-1423). Viralincorporation and display of CD4 was also demonstrated in recombinantherpes simplex virions constructed by inserting the CD4 gene into theHSV-1 genome under the control of a viral promoter (Dolter et al, 1993 JVirol 67 p189-195). However, nonviral proteins are generally excludedfrom assembling viral particles, or incorporated very inefficiently. Thepresent inventors believe a more reproducible strategy for the efficientincorporation and display of nonviral polypeptides on viruses which caninfect eukaryotic cells would be to fuse the nonviral polypeptide to aviral component, such as a viral spike glycoprotein, which carries asignal for incorporation into the viral particle.

The nucleic acid sequences encoding a nonviral peptide or polypeptidecan be linked, without disruption of the translational reading frame, tonucleic acid sequences coding for all or part of the gene III protein offilamentous bacteriophage, thereby creating a hybrid gene encoding achimaeric gene III protein (McCafferty et al, 1990 Nature 348 p552-554;Smith, 1985 Science 228 p1315-1317; Parmley and Smith, 1988 Gene 73p305-318; Scott and Smith, 1990 Science 249 p404-406). The details ofthe construction can be varied such that the gene III moiety of thechimaeric protein remains substantially intact or is lacking one or moredomains. When expressed in prokaryotic cells which are sheddingfilamentous bacteriophage, the chimaeric gene III protein isincorporated into a proportion of the progeny phage and those phagedisplay the chimaeric protein on their surface. Incorporation of thechimaeric protein is presumed to occur during phage assembly through thespecific interaction of the C-terminal gene III moiety of the chimaericprotein with other protein components of the phage particle. Correctfolding and function of the nonviral polypeptide moiety of theincorporated chimaeric gene III protein is apparent from the alteredbinding specificity of the phage particles, which corresponds with thebinding specificity of correctly folded nonviral polypeptide moiety. Itis possible to generate phage particles incorporating a combination ofwild type and chimaeric gene III protein or incorporating exclusivelythe chimaeric gene III protein.

However, bacteriophage do not infect eukaryotic cells and cannottherefore be employed as gene delivery vehicles for such cells. Nor canthey be used for the display of glycoproteins since their bacterialhosts lack the necessary enzymatic machinery for correct glycosylationof polypeptides.

In the case of nonenveloped viruses which can infect eukaryotic cells,initial binding to the target cell is mediated by the viral capsid whichis composed of a multimeric symmetrical array of virus-encoded capsidproteins. Short (up to 26 aminoacids) nonviral peptides have beendisplayed on the surface of nonenveloped polioviruses by replacingsurface-exposed polypeptide loops (up to 9 aminoacids) in the capsidproteins (Rose and Evans, 1991 Trends Biotech 9 p415-421). This wasachieved by genetic manipulation and subsequent transfection of afull-length infectious cDNA clone of the RNA genome. The display ofpeptides in poliovirus antigen chimaeras has been pursued with a view tousing the chimaeric particles as antigen presentation vehicles tostimulate immune responses against the displayed peptide in vaccinatedanimals or humans and for use as diagnostic reagents in serodiagnosis.Larger, functional, folded polypeptides have not been displayed on theouter surface of nonenveloped viruses which can infect eukaryotic cells.

In the case of all enveloped viruses and certain nonenveloped viruses(eg adenovirus) which can infect eukaryotic cells, initial binding tothe target cell is mediated by specialised multifunctional, oligomericspike glycoproteins rather than by simple unglycosylated monomericproteins such as the gene III protein of filamentous bacteriophage.Thus, it is not possible simply to extrapolate from the bacteriophagegene III display system in the design of chimaeric variants of theseoligomeric spike glycoproteins for incorporation into assembling virusparticles and display of functional nonviral polypeptides at the surfaceof the virion. Correct glycosylation and oligomerisation of the spikeglycoproteins of enveloped viruses is often required for successfultransport to the cell surface and incorporation into viral particles(Polonoff et al, 1982 J Biol Chem 257 p14023-14028; Enfield and Hunter,1988 Proc Natl Acad Sci USA 85 p8688-8692: Kreis and Lodish, 1986 Cell46 p929-937; Copeland et al, 1988 Cell 53 p197-209). Proteolyticcleavage of assembled oligomeric viral spike glycoproteins frequentlyoccurs during their transport through the Golgi apparatus to the cellsurface. Thus, trimeric Murine Leukaemia Virus envelope glycoproteinprecursors are proteolytically cleaved in the Golgi apparatus into p15TMtransmembrane and gp705SU surface components, and these components areheld together by noncovalent interactions or by covalent disulphidebonds (Pinter et al, 1978 Virology 91 p345-351).

Non-viral polypeptides have been displayed on a retrovirus by fusion tothe membrane anchor sequence of the retroviral spike glycoprotein.Adopting this strategy, incorporation of chimaeric CD4-envelope proteinswas demonstrated by immunoprecipitation of purified retroviral (RSV)particles, but there was no evidence for correct folding or function ofthe CD4 (Young et al, 1990 Science 250p1421-1423). Chimaeric VSV-Gproteins comprising the cytoplasmic and transmembrane anchor domains ofVSV-G spike glycoprotein fused to the ectodomain of CD4 wereincorporated into the envelopes of infectious VSV particles (Schubert etal, 1992 J Virol 66 p1579-1589). However, the authors were not able todemonstrate correct folding or function of the virally incorporated CD4and state that "Numerous experiments to demonstrate a specific tropismfor HIV envelope-expressing cells were not successful so far. ---- Inthe environment of a viral membrane, the receptor may not be functional".

The technique of insertional mutagenesis has been used to define domainsof the MoMLV genome which are amenable to small alterations withoutdeleterious effects on the virus (Lobel and Goff, 1984 Proc Natl AcadSci USA 81 p4149-4153). Viable linker insertions in the env gone of aninfectious molecular clone of MoMLV (eg in-6438-12 and in-7407-9) wereshown to generate infectious retroviruses whose spike glycoproteins hadpresumably incorporated the four-residue nonviral peptide encoded by theinserted linker. However, no attempt was made to demonstrate display ofsuch a peptide, nor was the possibility of surface display mentioned.

The present inventors have devised a novel strategy for theincorporation and display of nonviral polypeptides in fusion with viralglycoproteins, particularly oligomeric viral spike glycoproteins. Thenucleic acid sequences encoding a nonviral polypeptide are fused,without disruption of the translational reading frame, to nucleic acidsequences coding for the oligomeric viral spike glycoprotein. The hybridgene codes for a chimaeric glycoprotein in which the domain structureand organisation of the viral spike glycoprotein moiety remainsubstantially intact so as to conserve the post-translationalprocessing, oligomerisation, viral incorporation and, possibly,fusogenic activities. The nonviral polypeptide is fused close to theterminus of the mature spike glycoprotein which is known to be displayeddistally on the outside of the viral particle. To avoid possible sterichindrance between the nonviral polypeptide moieties which couldsignificantly inhibit oligomerisation, the chimaeric glycoprotein can beexpressed in virus-shedding cells in the presence of the wild-type virusspike glycoprotein such that each oligomeric unit need incorporate onlya single copy of the chimaeric glycoprotein. Adopting this strategy, wehave demonstrated incorporation of a chimaeric glycoprotein comprising asingle chain antibody fused to a retroviral spike glycoprotein intomurine ecotropic and amphotropic retroviral (MLV) particles. Moreover,in contrast to previous studies we have been able to demonstrate thatthe virally incorporated single chain antibody remains functional asevidenced by its ability to bind specifically to its target antigen(NIP).

A logical extension of these studies is the construction of vectors ofsimilar design for the display of nonviral peptides, polypeptides andglycopolypeptides other than single chain Fv antibody fragments onretroviral particles. Among the polypeptides and glycopolypeptidessuitable for display on the particles are Fv and Fab antibody fragments,T-cell receptors, cytokines, growth factors, enzymes, cellular adhesionproteins such as integrins and selectins, Fc receptors etc. There is noreason why two or more different nonviral polypeptides should not beincorporated into a single virus particle by their co-expression asfusion proteins in the same packaging cell. The display of nonviralpeptides, polypeptides or glycopolypeptides as similar fusions with theoligomeric spike glycoproteins of other retroviruses and with viruses ofother families also follows directly from the this invention. Animalviruses of the Adenovirus, Togavirus, Rhabdovirus, Paramyxovirus,Orthomyxovirus and Retrovirus families which have relativelywell-defined oligomeric spike glycoproteins are particularly suitablefor such manipulation.

The invention, in one aspect, thus provides a recombinant viral particlecapable of infecting eukaryotic cells, comprising a non viralpolypeptide fused to a substantially intact viral glycoprotein orchimera of viral glycoproteins and a displayed on the external surfaceof the particle.

The term "viral glycoprotein" means a glycoprotein encoded by a virus inits natural state. The viral glycoprotein is typically a viral spikeglycoprotein, i.e. a protein which in its natural state:

1. projects from the surface of the virus to be visible by electronmicroscope;

2. is oligomeric, having 2 to 6 subunits which may be identical or nonidentical, i.e. homo or heterooligomers;

3. is glycosylated;

4. comprises a structural signal which directs its efficientincorporation into the viral particle.

The chimera of viral glycoproteins must be capable of incorporation intothe viral particle to satisfy conditions 1 to 4 above. One example ofsuch a chimera is a protein comprising the transmembrane and cytoplasmicdomains of the trimeric Rous Sarcoma Virus envelope glycoprotein fusedto the trimeric extraviral domains of the influenza haemagglutinin (Donget al., 1992 J. Virol. 66, 7374-7382).

The virus is conveniently a retrovirus, but this is not critical.

The eukaryotic cells are typically mammalian, e.g. human.

The polypeptide and glycoprotein are fused together. i.e. in the form ofa single, continuous polypeptide chain.

It is important that the viral glycoprotein is substantially intact,i.e. retains all its domains, to conserve the post-translationalprocessing, oligomerisation, viral incorporation and, possibly,fusogenic activities. However, certain alterations, e.g. mutations,deletions, additions, can be made to the glycoprotein which do notsignificantly affect these functions, and glycoproteins with suchmodifications are considered substantially intact.

The non viral polypeptide is preferably fused close to the terminus ofthe mature glycoprotein which is known to be displayed distally on theoutside of the viral particle, so that folding of the distal terminaldomain is not significantly disturbed.

The non viral polypeptide generally comprises at least 6 amino acids,and may range from a short polypeptide to a fully functional protein.The polypeptide may be glycosylated. The polypeptide typically comprisesan antibody or antibody fragment, receptor, enzyme etc.

The non viral polypeptide can be selected to bind to a target eukaryoticcell, via a cell surface molecule, with the virus deliveringencapsidated nucleic acid to the target cell. The viral particles of theinvention thus find application in targeted gene delivery and targetedvirotherapy, as will be discussed in more detail below.

The non viral polypeptide conveniently comprises antibody or antibodyfragments e.g. heavy and light chain variable domains of an antibody,which may comprise framework regions homologous to the framework regionsof human antibodies. Such variable domains are conveniently derived fromphage display libraries, e.g. by being selected for binding to cellsurface molecules.

Antibody fragments of low immunogenicity and peptides with desirablebinding activities may be most versatile targeting agents forincorporation into particles for targeting human cells. Antibodyfragments could be expressed as single chain fragments (in which theheavy and light chain variable domains are located on the samepolypeptide, using by a flexible peptide linking the two domainsdirectly) or as two chain constructs, in which one chain is fused to thesecond polypeptide and the other is secreted and associates with thefusion protein. Other possibilities for targeting the virus to the cellsurface include cytokines, for example EGF for tumours with highexpression, or T-cell receptors cloned from tumour-specific T cells.

For targeted binding, for example to human tumour cells, the virusparticles should ideally not bind to other human cells. The non viralpolypeptide should therefore confer novel (e.g. antitumour) bindingactivity on the particle. It is therefore desirable to use viralparticles which do not naturally bind to the target cells, or which havebeen modified to destroy natural binding to the target cells. In thiscase, all binding to target cells will be attributable to the non viralpolypeptide.

The target cell specificity of such binding may be further enhancedwhere two or more novel (e.g. antitumour) binding activities aredisplayed on a single particle. The viral particle may thus include oneor more additional viral coat proteins, in addition to the fusionprotein. Such additional proteins may or may not bind to the targetcells and may or may not allow infection of the target cells. Additionalbinding activities conferred by the non viral polypeptide or viralprotein moiety of the fusion protein or by unmodified coat proteins maydecrease the specificity of binding to the tumour cells, and it willtherefore often be desirable to choose these proteins to give minimalbackground or to inactivate their binding activity, for example by sitedirected mutagenesis.

After virus binding, fusion with or translocation across the limitingmembrane of the target cell, sometimes preceded by endocytosis, is anecessary step which may require inclusion of specific (e.g. fusogenic)proteins in the coat of the viral particle (and these proteins shouldideally not increase the background binding to nontarget cells). Forexample, inclusion of fusogenic influenza haemagglutinin trimers,mutated to destroy sialic acid receptor binding activity, could triggerlow pH-dependent membrane fusion of endocytosed virus with the endosomalmembrane. Where the nonviral polypeptide is fused to a viral coatprotein, the coat protein moiety may itself carry the necessaryfusogenic or translocating capability.

Viruses which infect eukarvotic cells as genetic display packages

The isolation of genes encoding proteins with known binding propertieshas recently been facilitated by selection technologies utilisinggenetic display packages. The genes encoding the protein are packagedsuch that the encoded protein is displayed on the outside of thepackage. The package is then selected by its binding affinity (forexample an encoded antigen by binding to solid phase antibody), andreplicate. The tight linkage between genes and encoded protein allowpackages to be selected in rounds of binding and amplification, leadingto selection factors of more than one in a million. Replicable packageshave included mammalian cells--used to isolate the genes encodinglymphocyte surface markers from cDNA libraries (Strengelin S et al, 1988EMBO J 7 p1053-1059)--and filamentous bacteriophage--used to isolate thegenes encoding antibody fragments (McCafferty et al, 1990 Nature 348p552-554) and other proteins (Bass et al. 1990 Proteins: Struct FunctGenet 8 p309-314). The use of filamentous bacteriophage has even led tostrategies for building antibodies in bacteria and improving theirbinding affinities, and so by-passing immunisation (Marks et al, 1991 JMol Biol 222 p581-597; Hoogenboom and Winter, 1992 J Mol Biol 227p381-388).

Viruses which are capable of infecting eukaryotic cells have notpreviously been used as genetic display packages to facilitate selectiontechnologies. A typical DNA construct suitable for the generation andselection of a library of such genetic display packages should be smallenough to allow convenient manipulation and would preferably comprise:

(1) A eukaryotic cell expression cassette whose encoded polypeptidecomprises a structural signal to direct its efficient incorporation intoviral particles and a nonviral moiety (peptide or polypeptide) which isdisplayed at the surface of the virion. For certain of the novelselection strategies proposed below the polypeptide should not includedisplayed viral moieties which bind to receptors present on the chosentarget cells.

(2) Unique, noncomplementary restriction sites flanking the sequenceswhich encode the nonviral moiety to facilitate the cloning of a diverselibrary of peptide or polypeptide sequences into this site.

(3) An viral encapsidation signal sequence to direct the incorporationof the sequences encoding the displayed polypeptide into the viralparticles before they leave the packaging cell.

(4) Cis-acting sequences which, in the presence of appropriatetrans-acting factors, mediate amplification of the copy number of thenucleic acid sequences which code for the displayed polypeptide andassociated encapsidation signal sequence.

(5) A bacterial plasmid origin of replication and antibiotic resistancemarker gene to facilitate amplification of the plasmid in a bacterialhost strain (eg Escherichia coli).

In the case of a retroviral genetic display package, long terminalrepeat sequences, a tRNA primer binding site and a polypurine tractshould preferably be included to ensure reverse transcription andintegration of the encapsidated RNA in an infected target cell. It mayalso be desirable to include a selectable marker gene in theencapsidated nucleic acid to facilitate recovery of sequences encodingthe displayed polypeptide from infected target cells after a round ofselection.

DNA constructs encoding virally incorporated glycoproteins have notpreviously been constructed according to the format outlined above,presumably because the concept of using eukaryotic viruses as replicabledisplay packages to facilitate novel selection strategies has notpreviously been entertained (PCT/US89/03731).

Thus, viral encapsidation signal sequences were not included in theconstructs used to demonstrate viral incorporation of CD4 or CD4/viralenvelope chimaeric proteins into RSV or VSV particles (Young et al, 1990Science 250 p1421-1423; Schubert et al, 1992 J Virol 66 p1579-1589) andthe recombinant HSV-1 genome used to demonstrate inefficientincorporation of CD4 into HSV virions (Dolter et al, 1993 J Virol 67p189-195) is too large (170 kbp) for convenient manipulation.

The present inventors therefore designed and constructed the plasmidpNIPenv and have demonstrated that it exhibits all of the featuresnecessary for the generation of retroviral genetic display librarieswith which to demonstrate the selection strategies outlined below. Thus,murine ecotropic retroviral packaging cells transfected with theconstruct were shown to shed retroviral particles which:

(a) had incorporated the fusion polypeptide encoded by the constructcomprising the Moloney MLV (mouse ecotropic) envelope spike glycoproteinfused to a hapten-binding single chain antibody;

(b) bound specifically to the hapten recognised by the single chainantibody;

(c) encapsidated the nucleic acid sequences encoding the displayedfusion polypeptide;

(d) delivered the encapsidated nucleic acid to murine target cells,whereupon it was reverse transcribed, integrated and expressed;

(e) did not deliver the encapsidated nucleic acid to human target cells.

Moreover, sequences encoding the nonviral polypeptide moiety of thedisplayed fusion polypeptide were amplified and recovered from targetcells which had been infected by the retroviral genetic displaypackages. Murine amphotropic packaging cell lines expressing theconstruct were also shown to shed retroviral particles that hadincorporated the fusion polypeptide, bound specifically to the haptenNIP and were infectious for mammalian cells.

In another aspect, the present invention thus provides a recombinantviral particle capable of infecting eukaryotic cells, comprising a nonviral polypeptide coupled to at least part of a viral glycoprotein anddisplayed on the external surface of the particle; and nucleic acidencoding the non viral polypeptide and said at least part of a viralglycoprotein, the nucleic acid further comprising a packaging signalsequence.

In this aspect, the non viral polypeptide is coupled to at least pan ofa viral glycoprotein. The coupling may be by fusion, or may involve thetwo components being held together by short range covalent ornoncovalent forces.

Further, it is not necessary to use the entire glycoprotein, and part orparts may be removed or modified. Typically at least the cytoplasmic andtransmembrane components of the glycoprotein will be present. Theglycosylated regions of the glycoprotein need not be present, so thatthe glycoprotein in the form used may no longer be a glycoprotein. Theglycoprotein may have been modified, e.g. to destroy natural bindingproperties, for reasons as mentioned above.

A packaging signal sequence is a nucleic acid sequence recognised by acomponent of the viral particle and which mediates the inclusion of thenucleic acid in the viral particle. It is also known as an encapsidationsignal sequence.

Because the viral particles of this aspect include nucleic acid encodingthe non viral polypeptide and said at least part of a viralglycoprotein, the particles find application as replicating vectors forgene delivery, as will be discussed further below.

The nucleic acid may also encode structural and non-structural viralproteins which when expressed in infected target cells assemble into newinfectious viral particles. The new viral particles may incorporate thecoupled polypeptide and viral glycoprotein. The nucleic acid of the newviral particles may encode structural and non-structural viral proteinswhich on infection of target cells assemble into new infectious viralparticles which incorporate the coupled polypeptide and viralglycoprotein. These properties of viral particles that encapsidatenucleic encoding the coupled polypeptide and viral glycoproteindisplayed at their surface, could be critical for certain therapies. Forexample, a major limitation of protein-based targeted cancer therapiesis the inability of the protein to penetrate the tumour. Replicationcompetent viruses could overcome this penetration barrier since they canspread from cell to adjacent cell within the tumour. For this approachit would be desirable that the progeny viral particles are largelyretained within the tumour deposit and have high infectivity for tumourcells with low infectivity for normal host cells. Multivalent surfacedisplay of a tumour-specific antibody could facilitate this.

The nucleic acid of the viral particles may be engineered to contain asequence for transcription of an RNA product, or for expression of aprotein, by the infected cells. A whole range of proteins, peptides,antisense RNA transcripts and ribozyme sequences could be encoded withinthe virion for therapeutic effect, as illustrated by:

a) Targeted corrective gene replacement therapy for defects of genesencoding intracellular, cell surface or secreted proteins. For exampletargeted ex vivo or in vivo delivery of genes to correct the defect insickle cell anaemia or thalassaemia (globin genes to bone marrowprogenitor cells), alpha-1 antitrypsin deficiency (peptides to preventintracellular accumulation of mutant (Z) alpha-1 antitrypsin),haemophilia (factor VIII or factor IX genes to hepatocytes), familialhypercholesterolaemia (LDL receptor to hepatocytes).

b) Intracellular immunisation, for example targeted in vivo delivery (toCD4 expressing cells) of genes encoding proteins, antisense transcriptsor ribozymes which interrupt or abort HIV life cycle following virusentry.

c) Pharmacological gene addition, for example delivery of genes encodingtherapeutic antibodies, growth factors or cytokines to specific tissuesin vivo.

d) Cancer therapy. Delivery of genes encoding proteins which destroy thetarget cell (for example, a ribosomal toxin), indirectly stimulatedestruction of target cell by natural effector cells (for example,strong antigens to stimulate immune system) or convert a precursorsubstance to a toxic substance which destroys the target cell (forexample, a prodrug-activating enzyme). Encoded proteins could alsodestroy bystander tumour cells (for example with secreted antitumourantibody-ribosomal toxin fusion protein), indirectly stimulatedestruction of bystander tumour cells (for example cytokines tostimulate immune system or procoagulant proteins causing local vascularocclusion) or convert a precursor substance to a toxic substance whichdestroys bystander tumour cells (e.g. enzyme which activates prodrug todiffusible drug). Also, delivery of genes encoding antisense transcriptsor ribozymes which interfere with expression of cellular genes criticalfor tumour persistence (for example against aberrant myc transcripts inBurkitts lymphoma or against bcr-abl transcripts in chronic myeloidleukaemia).

The expression of any of the proteins encoded by the encapsulatednucleic acid can be regulated in known manner if required.

Regulation of gene expression by inclusion of appropriatetranscriptional promoter, enhancer, silencer or locus control sequenceswill be essential in many applications. For example, globin geneexpression must be correctly regulated for effective correction ofthalassaemia defects in haemoglobin production. For cancer therapy,tumour-specific gene expression (after targeted gene delivery) wouldenhance the overall targeting potential of the therapy. Improvedspecificity of gene expression in tumour cells could be achieved by useof naturally occurring, designed (modular, based on knowledge oftranscription factors present in a particular tumour) or randomlymutated and selected tissue-specific, differentiation-specific,inducible and/or transformation-sensitive control sequences to regulateexpression of genes encoded by the nucleic acid of the viral particles.

The viral particles of this aspect of the invention also findapplication in the generation of libraries of viral display packages.

Using a similar cloning strategy to that employed to generate pNIPenv,unique Sfi I and Not I restriction sites could be inserted, for examplebetween the codons for the 6th and 7th amino acids of the matureenvelope protein in a full-length infectious molecular clone of MoMLV(Shoemaker et al, 1980 Proc Natl Acad Sci USA 77 p3932-3936), withoutdisruption of the translational reading frame. After removing the Sfi Isite in the gag coding region of MoMLV (Shinnick et al, 1981 Nature 293p543-548), the vector could then be used to generate libraries ofreplication-competent retroviruses displaying variegated peptides,polypeptides or glycopolypeptides.

Another aspect of the invention thus provides a DNA construct suitablefor generation of a library of viral display packages, comprising a sitefor insertion of a sequence encoding a non viral polypeptide capable ofbeing coupled to at least pan of a glycoprotein and of being displayedon the external surface of a viral particle; and a suitable packagingsignal sequence.

The term viral display package is used to mean a recombinant viralparticle capable of infecting eukaryotic cells, comprising a non viralpolypeptide coupled to at least part of a glycoprotein and displayed onthe external surface of the particle, and nucleic acid encoding the nonviral polypeptide and said at least part of a viral glycoprotein.

The site for insertion conveniently comprises a cloning site, preferablyhaving 2 unique non complementary restriction sites.

The glycoprotein from which said at least part of a glycoprotein isderived is conveniently a viral glycoprotein, but this is not essentialand non viral or constructed glycoproteins having suitable propertiescan be used.

The construct may include a marker for selection of infected cells.

A logical extension of these studies is the construction of vectors ofsimilar design for the encapsidation of nucleic acid encoding adisplayed nonviral polypeptide on any virus of any family of viruseswhich can infect eukaryotic cells. The construction and use of similarvectors based on other virus families is particularly obvious forenveloped viruses of limited size and complexity such as VesicularStomatitis Virus, Influenza Virus, Semliki Forest Virus, Sindibis Virusand Sendai Virus which display oligomeric membrane spike glycoproteins.The construction and use of similar vectors derived from viruses of theAdenovirus family is also obvious since the adenovirus fibreglycoprotein has important features in common with the MoMLV envelopeglycoprotein in that it is a glycosylated homotrimer (Mullis et al, 1990J Virol 64 p5317-5323) Devaux et al, 1990 J Mol Biol 215 p567-588) whichis visible by electron microscopy as a spike projecting from the surfaceof the virion (Ruigrok et al, 1990 J Mol Biol 215 p589-596).

We envisage the use of such viruses in which the encapsidated nucleicacid codes for a modified viral spike glycoprotein in which non viralpeptides are inserted into, or substituted for surface loops of thespike glycoprotein, or in which domains of the spike glycoprotein amreplaced by nonviral peptides, polypeptides or glycopolypeptides, or inwhich non viral peptides, polypeptides or glycopolypeptides are insertedbetween domains of the viral spike glycoprotein or linked to itstermini. The encapsidated nucleic acid need not encode the displayedpeptide, polypeptide or glycopolypeptide as a fusion with a viral spikeglycoprotein, although this is preferred. We also envisage the use ofviruses in which the encapsidated nucleic acid encodes an engineeredderivative of a nonviral protein such as CD4 which may be efficientlyincorporated into the particles and which has been engineered to displaya non viral peptide, polypeptide or glycopolypeptide. We also envisagethe use of viruses in which the nonviral peptide, polypeptide orglycopolypeptide is anchored in the virus particle by fusion to asynthetic transmembrane polypeptide. Also envisaged is the use ofparticles encapsidating nucleic acid encoding bispecific diabodies(Holliger et al, 1993 Proc Nail Acad Sci USA 90 p6444-6448) or other"viral coat protein modifiers" as genetic display packages. The diabodywould act as a "viral coat protein modifier", binding at one end to thevirus and at the opposite end to the surface of a target cell. For thegeneration of libraries of such genetic display packages, the viral coatprotein modifier should bind with high affinity to the viral particlesbefore or during their release from the target cell, and should not beproduced in excess over the viral coat proteins

Generation of a library of genetic display packages which can infecteukarvotic cells

The unique noncomplementary restriction sites (Sfi I and Not I) inpNIPenv which flank the sequences encoding the nonviral polypeptide(single chain antibody) moiety of the displayed protein were chosen tofacilitate the rapid cloning of any PCR-generated library of DNAfragments into this site and to facilitate the direct shuttling of DNAfragments from pre-selected phage antibody libraries incorporating thesame flanking restriction sites. Clearly, there are alternativecombinations of noncomplementary restriction sites which would serveequally well and in some cases (the insertion of random sequencesencoding short peptides, for example) a single restriction site shouldshould be sufficient.

The generation of a diverse library of recombinant retroviruses has beendemonstrated previously by transient transfection of retroviral plasmidsinto retroviral packaging cells (Murphy and Efstratiadis, 1987 Proc NatlAcad Sci USA 84 p8277-8281). The library size is limited by theefficiency of plasmid transfection, first into E. coli for growth andpurification of the plasmid ligation product and second into theretroviral packaging cells for generation of the retroviral geneticdisplay library. Employing recently developed and highly efficientmethods of gene delivery to mammalian cells (Curiel et al, 1992 Hum GeneTher 3 p147-154), we estimate that a library size of 10⁸ retroviralgenetic display packages should be possible.

It is proposed to use this approach to generate libraries of recombinantviruses whose members encapsidate nucleic acid sequences encoding thenonviral peptides, polypeptides or glycopolypeptides which are displayedat their surfaces. There are a number of post-translationalmodifications to polypeptides which occur uniquely in eukaryotic cells,are not possible in prokaryotic host cells and are required for correctfolding and function of the polypeptides. The generation of librariesdisplaying such polypeptides (glycoproteins, for example) is thereforenot possible using prokaryotic genetic display packages such as thebacteriophage display system, but is possible using eukaryotic displaypackages as disclosed in this invention.

Libraries can be produced of a wide range of peptides and glycopeptides,e.g. antibodies, antibody fragments such as single chain antibodies, Tcell receptors, growth factors, adhesins, selectins etc.

In a further aspect the present invention thus provides a library ofviral display packages prepared using the DNA construct of theinvention.

Novel selection strategies

Variegated libraries of recombinant viruses which encapsidate nucleicacid encoding non viral peptides, polypeptides or glycopolypeptidesdisplayed at their surface provide the basis for novel selectionstrategies. Previously, using phage display libraries, various selectionstrategies have been employed to select and isolate members of thelibrary on the basis of the binding specificity, binding affinity, orcatalytic activity of the displayed polypeptide. However, phage displaylibraries do not provide for methods which select for the ability of adisplayed polypeptide to enhance delivery of nucleic acid into aeukaryotic target cell. Nor has the concept of such an application for alibrary of replicable display packages been previously disclosed.

The efficiency of delivery of nucleic acid into a target cell by a virusis influenced by the specificity and affinity of the initial interactionbetween the virus and the target cell surface. The present inventiontherefore provides for novel methods of selection (based on the presenceof the viral nucleic acid in the infected target cell) to isolatenucleic acid sequences encoding polypeptides which, when displayed onthe surface of a recombinant virus, can increase the efficiency withwhich the virus delivers its encapsidated nucleic acid to the interiorof a eukaryotic target cell, either in a cell-specific or anon-cell-specific manner.

The fate of the nucleic acid delivered to a target cell by a virus(intracellular transport, genome conversion, integration, amplification,gene expression) is influenced by the infectious entry pathway which inturn is influenced by the specificity of the initial interaction betweenthe virus and the target cell surface (Goud et al, 1988 Virology 163p251-254, for example). The invention therefore provides for novelmethods of selection (based on the intracellular localisation,conversion from RNA to DNA, integration, amplification or expression ofthe viral nucleic acid in the infected target cell) to isolate nucleicacid sequences encoding polypeptides which, when displayed on thesurface of a recombinant virus, can enhance the efficiency of deliveryof the encapsidated nucleic acid to the interior of a eukaryotic targetcell via a non-abortive infectious entry pathway.

The fate of the nucleic acid delivered to a target cell by a virus isalso influenced by host cell factors (such as the state of activation ofthe host cell or its position in the cell cycle) which themselves can beinfluenced by signalling molecules which interact with receptors on thesurface of the target cell (Springett et al, 1989 J Virol 63 p3865-3869;Harel et al, 1981 Virology 110 p202-207). The invention thereforeprovides for novel methods of selection (based on the intracellularlocalisation, conversion from RNA to DNA, integration, amplification orexpression of the viral nucleic acid in the infected target cell) toisolate nucleic acid sequences encoding polypeptides which, whendisplayed on the surface of a recombinant virus, can transmit a signalto the target cell which leads to alterations in the interior of thetarget cell that regulate the fate of the delivered nucleic acid, e.g.expression thereof.

The experimental parameters which are amenable to manipulation and whichmay affect the outcome of such selection procedures include:

1. The underlying composition of the viruses in the library which is inturn determined by the design of the vector plasmid and packaging systemused to generate the library

The nucleic acid encapsidated in the viruses may be nondefective (iecompetent for the production of infectious progeny viruses in infectedtarget cells) or defective and may or may not include selectable markergenes (conferring antibiotic resistance, for example).

The surface composition of the viruses in the library (and hence theirstarting host range properties) is determined initially by the choicethe packaging system. For example, by appropriate choice of packagingcells, the RNA transcript and encoded fusion protein of apNIPenv-derived library can be incorporated into retroviruses which alsodisplay unmodified mouse ecotropic MLV envelope proteins, unmodifiedmouse amphotropic MLV envelope proteins, or which do not displayunmodified envelope proteins.

The starting host range of the viruses in the library may be furtherdetermined by the nature of the viral moieties of the modified spikeglycoproteins, employed to anchor the displayed nonviral polypeptides inthe viruses.

2. The composition of the library of displayed polypeptides

This may comprise a diverse collection of peptides, antibody fragments(Fv, scFv, Fab), T-cell receptors, variants of a growth factor,cytokine, viral protein or enzyme generated by random mutagenesis, acDNA expression library etc. The library may be derived from a largerphage display library pre-selected for a specific bidding activity orcatalytic activity and subcloned into the viral display vector.

The size of the library (ie diversity, number of viruses displaying aunique nonviral polypeptide) is determined by the methods used togenerate diversity in the vector inserts encoding the displayedpolypeptides, the efficiency of their introduction into the vectorcloning site, the efficiency of the introduction of the vector into E.coli and the efficiency of introduction of the vector plasmid libraryinto the packaging cells. There are numerous methods available togenerate diversity in a library of viruses, and these have beendescribed previously in relation to phage antibody libraries. TheIox-cre, or other recombinase systems which are active in eukaryoticcells may be employed to further increase the diversity of the libraryby the random recombination of components of the vector inserts.

The final virus titre in the library will be a product of the diversityof the library and the number of copies of each species in the library.Titre is a function of the efficiency of the packaging system, whichrelates to the efficiency of expression of the vector and helperfunctions (ie viral structural and nonstructural proteins), and to theefficiency of amplification and encapsidation of the vector genome inthe packaging cells. The virus titre could therefore be enhanced, forexample, by the use of high copy number episomally replicating plasmidsand/or strong cellular promoter/enhancers and by the appropriateselection of highly efficient encapsidation signal sequences.

3. Pre-adsorption of the library

Pre-adsorption of the library of viruses by its application to a surfacecoated with purified antigen or by its application to nontarget cellscan be used to deplete the library of viruses with unwanted bindingspecificities.

The choice of antigens or nontarget cells for pre-adsorption conditionsselected for the pre-adsorption step (time, temperature, pH, compositionof medium, presence of blocking proteins etc) may significantlyinfluence the outcome of the selection procedure.

4. The composition of the target cells

The species of origin, tissue of origin, state of differentiation, stateof activation, state of synchrony and proliferative status of the targetcells are important variables which will influence the outcome of theselection strategy.

The absolute number and purity of the eukaryotic target cells can alsobe varied.

There may be circumstances in which the target cells remain in theirnatural environment in a living organism or are artificially implantedinto a living organism.

5. The conditions under which the library of viruses is contacted withthe target cells

The time, temperature, pH, composition of medium, presence of competitorantigen or blocking proteins ere may significantly influence the outcomeof the selection procedure.

The target cells may alternatively be contacted with the library ofvirus-producing cells.

The library may be contacted with target cells in a living organism bythe direct inoculation (by any route) of the vector library, the libraryof viruses or a library of virus-producing cells.

6. The treatment of the target cells subsequent to their exposure to thevirus library

The target cells may be maintained in tissue culture (or in a livingorganism) for a variable period of time without further selection.

A pure population of target cells may be selected from a mixedpopulation of target cells, for example by fluorescent staining andfluorescence-activated cell sorting.

The target cells may be stimulated, for example by the application ofselected growth factors or cytokines.

The target cells may be selected on the basis of their expression of thedelivered viral nucleic acid. If the viral nucleic acid encodes anantibiotic resistance marker such as neomycin phosphotransferase,exposure of the cells to the antibiotic G418 will select for those cellsexpressing the viral nucleic acid. The viral nucleic acid sequences canbe recovered from the infected target cell population before or after ithas been subjected to a further selection process to eliminate thosecells which were not successfully infected or which do not express theprotein(s) encoded by the delivered nucleic acid. Alternatively, thetarget cell population could be stained with fluorescent antibodiesagainst the viral component of the modified spike glycoprotein (MoMLVenvelope glycoprotein, for example) encoded by the viral nucleic acidand positively staining cells could be isolated byfluorescence-activated cell sorting (FACS). FACS would also facilitatethe selection of cells on the basis of the level of expression of thetransferred nucleic acid. Fluorescent staining of cells for expressionof the MoMLV envelope spike glycoprotein has previously beendemonstrated (Chesebro et al, 1981 Virology 112 p131-144).

7. The mechanism of recovery from the infected target cells of thedelivered nucleic acid which encodes the displayed polypeptide

The recovery of the nucleic acid sequences encoding the virus-displayednonviral peptide, polypeptide or glycopolypeptide after the delivery ofthe nucleic acid to the a target cell can be achieved by PCRamplification using flanking oligonucleotide primers as demonstrated inexample 1. PCR amplification may be from whole cells, from highmolecular weight DNA or low molecular weight DNA extracted from thecells or from cDNA prepared from RNA extracted from the cells.

Alternatively, the viral nucleic acid could be amplified and recovereddirectly from the infected target cells into progeny viruses bysuperinfection with wild-type virus, by transfection of a suitablehelper plasmid (encoding the retroviral gag and pot proteins if usingthe vectors disclosed in example 1), or by using a library ofrecombinant viruses encapsidating a full-length infectious viral genomeinclusive of the sequences encoding the displayed polypeptide.

8. Number of rounds of selection

After recovery of the viral nucleic acid encoding the displayed nonviralpolypeptide, it may be desirable to to use them to create a Secondarylibrary of viruses for further rounds of selection. This could beachieved by digesting the PCR amplification product with appropriaterestriction enzymes (Sfi I and Not I if using the vectors disclosed inexample 1) and cloning the digested product into the original vector.There is no limit to the possible number of rounds of selection.

9. Diversification of the product of previous rounds of selection andthe generation of secondary libraries for subsequent rounds of selection

Such diversification between successive rounds of selection provides forthe directed evolution of the displayed polypeptides towards a desiredbiological function (Joyce, December 1992 Scientific American p48-55).

Various methods may be employed for the diversification-of the nucleicacid sequences derived from a round of selection and the similarapplication of many such methods has been demonstrated previously usingphage display libraries.

The high spontaneous mutation rates of certain RNA viruses (eg Leider etal, | 988 J. Virol. 62 p3084-3089) and their possible enhancement mayalso provide for novel means by which to diversify the library betweenrounds of selection.

Retrovirus display packages could be used for applications analagous tothose which have been developed for filamentous bacteriophage. Thus,retrovirus display libraries could be selected directly on anantigen-coated solid support or indirectly with soluble biotinylatedantigen followed by capture on a streptavidin-coated solid support.Bound virus could be amplified by infection of a retrovirus packagingcell line. Ecotropic virus cannot infect ecotropic packaging cells andamphotropic virus cannot infect amphotropic packaging cells due to thephenomenon of superinfection resistance. However, ecotropic virus caninfect amphotropic packaging cells and vice versa. It should thereforebe possible to amplify bound virus after each round of selection byinfection of the appropriate (ecotropic or amphotropic) packaging cellline. The theoretical maximum achievable retrovirus display library sizedoes not compare favourably with the theoretical maximum size of abacteriophage display library. It is therefore unlikely that retrovirusdisplay libraries will challenge the established applications of phagedisplay libraries such as in vitro antibody selection and affinitymaturation.

Retrovirus display libraries may facilitate selection of proteins whichrequire post-translational modifications for full activity. Many of theproteins manufactured in mammalian cells (particularly cell surface andsecreted proteins) are subject to post-translational modifications suchas glycosylation or proteolytic cleavage. These post-translationalmodifications, which may be critical for the functional activity of themature protein, do not occur in E. coli or other prokaryotic expressionsystems. There is thus a group of proteins which are not amenable tofunctional selection in a phage display library because of theirrequirement for post-translational modifications which are possible onlyin a mammalian cell expression system. Such proteins should be amenableto selection in a retrovirus display library.

See also WO92/01047.

Potential applications in the field of therapeutic gene transfer

1. In parallel with advances in the safety and efficiency oftechnologies for the transfer of genes into human cells, either ex vivoor in vivo, an era of experimental human gene therapy has begun. Genetherapy strategies have been proposed for many human diseases, includingrare heritable genetic defects of which there are more than 4000 andmany common diseases including cancer, AIDS, hypertension, atheroma anddiabetes (Anderson, 1992 Science 256 p808-813; Friedmann, 1992 NatureGenet 2 p93-98; Russell, 1993 Cancer J 6 p21-25). The current inventiontherefore has potentially important application in almost every area ofhuman medicine.

Replication-defective viruses displaying antibodies and other nonviralpeptides, polypeptides or glycopolypeptides and encapsidating genesencoding therapeutic products (eg proteins, ribozymes, antisense RNA)could be used to achieve efficient and selective delivery and expressionof the encapsidated genes to target cells (which may be stem cells,differentiated cells or transformed cells of any tissue of origin), tostimulate the target cell to divide or to enter a specific programme ofdifferentiation at the time of contact between virus and target cell, orfor virus purification on a solid support coated with an antigen whichbinds to an altered surface component of the virus.

For example, ex vivo gene delivery, to human haemopoietic stem cells(HSCs) is inefficient using amphotroic retroviral vectors, a problemwhich is in pan due to the quiescence of the HSCs but may also reflect alow density of specific receptors for amphotropic retroviruses. Thus, amodified retrovirus could be assembled to include displayed polypeptideswhich, singly or in combination-on the same particle, could allow thepurification of the recombinant retroviral particles, bind themselectively to the HSCs, enhance the efficiency of viral entry into theHSCs and trigger division of the HSCs or otherwise enhance theefficiency of gene delivery and expression in the stem cells. The HSCsmay be contacted with the recombinant viruses by direct exposure to thevirus or by co-cultivation with the cells producing the recombinantretroviruses.

For the genetic modification of haemopoietic stem cells (HSCs) by directin vivo gene transfer, the recombinant retroviruses should ideally havethe following properties:

They should be easy to produce and easy to purify.

They should be capable of gaining access to their intended target cellpopulation. The bone marrow compartment, which houses the HSCs,possesses a sinusoidal circulation and should therefore be accessible toretroviral particles delivered into the bloodstream. The retroviralparticles should persist intact in the bloodstream until they aredelivered to the bone marrow compartment and interact with their targetcells and should therefore be resistant to complement and otherpotentially virolytic factors in the bloodstream or interstitial fluid.They should neither bind nor infect nontarget cells which could lead totheir premature sequestration in nontarget tissues.

Having gained access to the target cells, they should selectively bindto a cell surface receptor or receptors, present on HSCs but absent fromnontarget cells, and this should be followed by the fusion of theretroviral envelope with the plasma membrane of the HSC and the deliveryof the retroviral core particle and its encapsidated nucleic acid intothe cytoplasm of the HSC.

Since most HSCs remain quiescent for long periods of time, and sinceretroviral reverse transcription and proviral integration proceedinefficiently in the absence of cell division, the retrovirus shouldalso stimulate the target cells to divide at, or shortly after, the timeat which it delivers its nucleic acid to the target cell.

The provirus should integrate efficiently into a predetermined site in ahost cell chromosome where it does not influence the structure orexpression of the genes of the host cell by cis-acting mechanisms.

The regulatory elements contained within the retroviral provirus whichcontrol the expression of the proteins encoded by the provirus shouldcause those proteins to be expressed at the appropriate time and at thedesired level in the HSCs or in their differentiated progeny in alineage-specific manner.

When expressed at the desired level in the genetically modified HSCs orin their progeny, the proteins encoded by the retroviral provirus shouldconfer a therapeutically beneficial phenotype on the cells.

The altered viral surface components of this invention which can beintelligently chosen (CD34, stem cell factor), selected from virusdisplay libraries or evolved by repeated selection and diversificationof virus display libraries could therefore be incorporated into viralparticles exhibiting any or all of the other desirable featuresmentioned above to further improve the efficiency and selectivity ofgone delivery to HSCs in vivo.

Similar considerations apply to stem cells, differentiated cells andtransformed cells of any tissue of origin.

2. Replicating viruses displaying antibodies or other nonviral peptides,polypeptides or glycopolypeptides could be used for targetedvirotherapy. For example a virus displaying a nonviral moiety mediatinginfection via the lymphoma idiotype of a B cell lymphoma(tumour-specific antigen) might spread from cell to cell through atumour deposit without infecting normal host cells. Used in conjunctionwith B-cell specific regulatory elements (promoters/enhancers)incorporated into the viral nucleic acid to restrict the expression ofthe viral genome to cells of B-lineage, it might be possible to generatea virus highly specific for the B cell lymphoma. The viral genome couldbe delivered to initiate the infection by direct intratumoural orsystemic inoculation of a DNA construct encoding the virus, the virusitself or the cells producing the virus. The viral surface component orcomponents mediating selective infection of the human lymphoma cellsmight be selected from a virus display library, generated from a phageantibody library pre-selected on the lymhoma cells or on purifiedidiotypic immunoglobulin derived from the lymphoma, and might be furtherevolved by repeated rounds of diversification and selection.

3. The nonviral peptides, polypeptides or glycopolypeptides isolated bythe novel selection strategies of this invention could be used as usefulcomponents of replication defective viral gene delivery vehicles or ofreplication-competent viral vectors.

They may also be useful as components of any gene delivery vehicle (egliposome, virosome, directly conjugated to DNA, physically linked tosurface of preformed virus) to enhance the selectivity and efficiency ofgene delivery to target cells.

They may also be used to purify and clone the cellular genes encodingthe cell surface proteins to which they bind.

They may also be used as therapeutic or diagnostic protein reagents oras components of such reagents for in vivo tumour targeting, tumourimaging, immunohistochemistry etc.

Viral particles in accordance with the invention may be produced ex vivoby introducing a vector containing appropriate nucleic acid intoeukaryotic cells, and culturing the cells to produce viral particles.Transduced cells are selected from non-transduced cells, and virusparticles may be isolated from the culture medium. e.g. by affinitychromatography.

Viral particles in accordance with the invention may also be produced invivo by introducing a vector containing appropriate nucleic acid intoeukaryotic cells in vivo.

Direct inoculation of vector (naked, coated or encapsulated) into tumourdeposits, for example, would be followed by cellular uptake andexpression with subsequent production of viral particles displaying atumour-binding fusion protein. This could be the simplest way toestablish virus production in vivo for the targeted virotherapy approachto cancer and may be more efficient than direct inoculation of virus forother targeted gone therapy strategies, provided that thevirus-producing cells expire naturally or can be easily destroyed aftera suitable time.

The viral particles of the invention may be used to infect eukaryoticcells in vivo or ex vivo by suitable administration of the particles.Direct injection of viable or killed virus-producing cells into tumourdeposits may have advantages of simplicity compared to other methods ofdelivery, particularly with regard to tumour deposits, where the goal isselective destruction of the target tissue.

Possible applications of viruses displaying peptides, e.g. antibodyfragments

1. Host range modification

Recombinant retroviruses are used for efficient transfer of non viralgenes to a variety of target cells. Amphotropic retroviruses bind to areceptor which is present on most mammalian cells and are therefore usedfor gone transfer to human target cells. Ecotropic retroviruses bind toa receptor which is present on murine and some other rodent cells, butis absent from human cells.

The host range of an ecotropic retrovirus might be extended in a highlyspecific way by display of an antibody against a cellular receptorpresent on a specialised subset of human cells. For example, recombinant(`ecotropic`) retroviruses displaying an antibody (or growth factor orpeptide) against a receptor present on human haematopoietic progenitorcells (CD34 or stem cell factor, for example) might be used for targetedgene delivery to these cells, either ex vivo by incubatingunfractionated bone marrow with virus, or in vivo by intravenousdelivery of virus. For in vivo delivery, other modifications to thevirus particles would be necessary to reduce their sensitivity to humancomplement.

An antibody displayed on an amphotropic retrovirus could be used as atissue retention signal. For example, genes can be delivered into tumourdeposits by direct inoculation of retroviruses or retrovirus producercells. Virus particles displaying an antitumour antibody might beretained more efficiently at the site of inoculation.

An antibody displayed on an amphotropic retrovirus could be used toenhance its infectivity for a specific human target cell. For example,amphotropic retroviruses displaying anti-CD34 antibodies might transfertheir genes more efficiently to human haematopoietic progenitor cells.This effect could be mediated through preferential localisation of viruson CD34 positive cells and by slowed dissociation of virus from targetcell because of the high affinity multivalent interaction between CD34on the target cell and anti-CD34 antibody on the virus.

2. Target cell priming

Binding, fusion and reverse transcription of the viral genome are notdependent on target cell proliferation. However, integration of thereverse transcribed viral genome and subsequent viral gene expression donot proceed unless the target cell enters S phase some short time afterit has been infected. This requirement seriously limits the efficiencyof gene transfer into noncycling or slowly cycling target cellpopulations. Thus, ex vivo gene transfer efficiencies to haematopoieticprogenitor cells have tended to be low. If the virus particles were todisplay antibody fragments, peptides or other ligands which couldstimulate the target cells to divide at the time of virus entry., theefficiency of provirus integration in the stimulated target cells mightbe significantly enhanced.

3. Virus purification and concentration

Recombinant retroviruses are difficult to purify and to concentrate.Viruses displaying functional antibody fragments or other ligands (e.g.short peptides) can be extracted from cell culture supernatant by virtueof their ability to bind to an antigen- or receptor-coated solidsupport. It should also be possible to elute the bound virus particleswithout damaging their integrity and infectivity. This will facilitatepurification and concentration of the recombinant viruses which will beuseful for ex vivo gene delivery and essential for in vivo genedelivery.

4. Selection of antibodies, ligands or peptides which bind to cellsurface receptors

Viruses displaying peptides e.g. antibody fragments have potentialapplications as display packages which encapsidate the nucleic acidsequence coding for the non viral protein displayed on the particlesurface. The linkage between nucleic acid coding sequence and encodedprotein or peptide provides a basis for generation of virus displaylibraries from which it will be possible to select antibodies or otherligands with desired binding specificities and binding kinetics. Thelibrary size will be limited by the efficiency and scale with whichplasmid DNA can be transfected into retrovirus packaging cells. 10⁸members is a suggested upper limit.

A virus display library composed of ecotropic virus particles displayingantibodies, peptides or other ligands against human cell surfacedeterminants could be screened for ability to infect human cells. Onlythose particles binding to appropriate human cell surface structuresshould be capable of delivering their nucleic acid to the human targetcells. To facilitate this type of screening it will be necessary to linkthe nucleic acid sequence for the displayed protein to a selectablemarker gene such that both are encapsidated within the viral particle.Human cells infected by recombinant `ecotropic` particles could then beselected for expression of the marker gene. It would then be a simplematter to isolate from these cells (by PCR for example) the gene codingfor the protein which was displayed on the original infectious viralparticle. This strategy would represent a new approach to the functionalselection of antibodies or other ligands against cell surface receptors.

5. Evolution of new virus tropisms

In the strategy outlined above, the integrated provirus derived from theinfecting particle is rescued by PCR. An alternative would be tointroduce further rounds of amplification and selection prior to resueof the provirus. Amplification could be achieved by using (as the targetfor infection with the retrovirus display library) a human cell lineexpressing retroviral gag, pol and ecotropic env proteins. Onceinfected, human target cells of this nature would generate replicacopies of the invading retrovirus particle for use in subsequent roundsof selection on the same cells. This would also permit rescue ofantibody V genes (or other sequences fused to the retroviral envelopegene) directly from progeny virions by PCR of (reverse transcribed)viral nucleic acid. Alternatively, it might be possible to generate alibrary of retroviral display packages capable of autonomousreplication. This could be achieved by inserting the Sfi/Not cloningsite into the env gene of a wild type retroviral genome, but would bedependent on the ability of the envelope fusion protein to form stableoligomers which are displayed on the viral surface in the absence ofwild type envelope protein and retain the ability to mediate thepost-binding events which lead to fusion of virus envelope with targetcell membrane. It is possible that oligomerisation of antibody-envelopefusion protein protomers is prevented by steric hindrance between theantibody domains of adjacent protomers. Short peptides, or even singledomain proteins displayed at the same site would be less likely tohinder oligomerisation by virtue of their smaller size.

The natural (high) mutation rate of a retrovirus relates to reversetranscriptase errors and recombinations between co-packaged viralgenomes during the process of reverse transcription. In conjunction withthe selection and amplification strategies outlined above, thesenaturally occurring mutations may allow the evolution of new virustropisms since they will lead to alterations in the fine specificity andaffinity of the antibody or ligand displayed on the surface of thevirions. In this way, it might be possible, for example, to generateviruses with evolved tropisms for specific types of cancer cell and touse such viruses for cancer therapy.

6. Antigen-selection of retroviruses displaying antibodies

Besides the selection strategies outlined above, retrovirus displaypackages could be used for applications analagous to those which havebeen developed for filamentous bacteriophage. Thus, retrovirus displaylibraries could be selected directly on an antigen-coated solid supportor indirectly with soluble biotinylated antigen followed by capture on astreptavidin-coated solid support. Bound virus could be amplified byinfection of a retrovirus packaging cell line. Ecotropic virus cannotinfect ecotropic packaging cells and amphototropic virus cannot infectamphotropic packaging cells due to the phenomenon of superinfectionresistance. However, ecotropic virus can infect amphotropic packagingcells and vice versa. It should therefore be possible to amplify boundvirus after each round of selection by infection of the appropriate(ecotropic or amphotropic) packaging cell line. The theoretical maximumachievable retrovirus display library size does not compare favourablywith the theoretical maximum size of a bacteriophage display library. Itis therefore unlikely that retrovirus display libraries will challengethe established applications of phage display libraries such as in vitroantibody selection and affinity maturation.

Virus display libraries may facilitate selection of proteins whichrequire post-translational modifications for full activity. Many of theproteins manufactured in mammalian cells (particularly cell surface andsecreted proteins) are subject to post-translational modifications suchas glycosylation or proteolytic cleavage. These post-translationalmodifications, which may be critical for the functional activity of themature protein, do not occur in E. Coli or other prokaryotic expressionsystems. There is thus a group of proteins which are not amenable tofunctional selection in a phage display library because of theirrequirement for post-translational modifications which are possible onlyin a mammalian cell expression system. Such proteins should be amenableto selection in a virus display library.

7. Viruses displaying other polypeptides

Besides the display of functional antibody fragments on viral particles,one can envisage the display of a variety of other ligands in fusionwith the viral envelope protein. Peptides, growth factors, cytokines,integrins, adhesins, selectins and T cell receptors are examples ofligands which could be displayed. Additionally, by analogy withfilamentous bacteriophage, it would be possible to display functionalenzymes on the surface of viral particles.

8. Viruses displaying multiple non viral polypeptides

In principle, there is no reason why two different envelope fusionproteins should not be simultaneously expressed in a packaging cell andincorporated into one viral particle. This might be useful in a numberof situations. For example, it may be desirable to generate particlesdisplaying one polypeptide to mediate binding to the target cell andanother polypeptide to stimulate the target cell to divide.Alternatively, a virus particle displaying two distinct polypeptides mayexhibit greater specificity for target cells expressing both of thecorresponding receptors than for target cells expressing only onereceptor.

9. Other enveloped viruses displaying non viral polypeptides

The electron micrograph appearances of many enveloped viruses reveal thepresence of surface structures which can best be described as `knobs onspikes`. More detailed comparison of certain unrelated viruses suggeststhat these EM-visible surface structures have a number of features incommon. They are frequently synthesised as a polyprotein precursor whichis subjected to extensive post-translational modification during itstransit to the surface of the cell in which it was synthesised. N-linkedglycosylation which occurs in the endoplasmic reticulum appears to benecessary for correct folding of the polyprotein precursor and this isfollowed by the formation of oligomers (often trimers) which aretransported into the Golgi compartment. Protomers are proteolyticallycleaved in the Golgi into a membrane-anchored (transmembrane) andluminal (surface) component. This cleavage does not lead to disruptionof the oligomeric structures since the surface and transmembranecomponents remain (covalently or noncovalently) associated, with the newhydrophobic N-terminus of the transmembrane components buried in thecentre of the oligomeric structure. After additional carbohydrateprocessing, the oligomeric structure arrives at the cell surface and isincorporated into budding viral particles. Studies of influenzahaemagglutinin (HA), which conforms to the pattern outlined above,indicate that docking to the target cell membrane is followed by aconformational change in the HA trimer leading to exposure of the buriedhydrophobic N-termini of the transmembrane component. These exposedhydrophobic sequences are thought to initiate the fusion of virusenvelope and target cell membrane by penetrating the lipid membrane ofthe target cell.

Thus, despite the lack of sequence homology between the envelopeproteins of unrelated viruses, there is good reason to believe that theyadopt similar three-dimensional structures and utilise similarmechanisms for docking and fusion with the target cell membrane. Allmammalian viruses whose envelope proteins conform to this pattern(examples include other retroviruses, orthomyxoviruses andparamyxoviruses) should therefore be amenable for surface display of nonviral polypeptides according to the strategy which we have successfullyadopted for Moloney MLV. Possible advantages offered by other virusesinclude higher virus titres, higher mutation or recombination rates,ability to use different cell surface molecules as surrogate receptors,ease and speed of virus production, higher density and absolute numberof nonviral polypeptides incorporated per virion.

10. Targeted Gene delivery

Replication-defective viruses displaying antibodies and other non viralpeptides, polypeptides or glycopolypeptides can be used for targetedgene delivery, increased efficiency of gene delivery, stimulation ofcell division at time of contact between virus and target cell, viruspurification. A good example would be using human haemopoietic stem cellas a target. Ex vivo gene delivery to these cells requires that they bepurified and stimulated to divide, and occurs at low efficiency withretroviral vectors (which do better than any other vector). Modifiedvirus could include displayed polypeptides which, singly or incombination, target the stem cells (without the need for purification),trigger division of the stem cells (with no need for stimulation) orotherwise enhance the efficiency of gene delivery to stem cells. In vivogene delivery to these cells requires efficient targeting andstimulation of division of target cell. One could deliver virus as DNAconstructs, virus or as virus-producing cells.

11. Targeted Virotherapy

Replicating viruses displaying antibodies and other non viral peptides,polypeptides or glycopolypeptides can be used for targeted virotherapy.A good example would be treatment of B-cell lymphoma, using a viruswhich displays a non viral moiety mediating infection via the lymphomaidiotype (tumour-specific antigen). The virus would spread through thetumour deposit without infecting normal host cells. Use in conjunctionwith B-cell specific promoters/enhancers, should give highly specificvirus. Again, one could deliver a virus as DNA construct, virus orvirus-producing cells.

12. Novel Selection Strategies

Non viral peptides, polypeptides or glycopolypeptides isolated by novelselection strategies can be used:

(a) As virus components for applications outlined in 10 and 11 above.

(b) As components of any gene delivery vehicle (e.g. liposome, virosome,directly conjugated to DNA, physically linked to surface of preformedvirus).

(c) As therapeutic or diagnostic protein reagents (e.g. tumour-targetingreagent).

(d) As components of such therapeutic or diagnostic reagents.

(e) As a means to clone the genes encoding the cell surface componentsto which they bind and subsequent use of such cell surface components.

The encoded protein could alternatively be a viral coat protein modifiersuch as a bispecific (anti-virus, anti-X) diabody, which associates withthe virus before or immediately after it is released from the cell suchthat anti-X moiety is displayed on the outide of the virion.

13. Gene Therapy

Viral vectors displaying non viral proteins or peptides may also offer avehicle for somatic gene therapy (Friedmann, 1989, Science, 244,1275-1281). Retroviruses can transfer genes efficiently to cells butthey deliver their therapeutic genes to both target and nontarget cells,necessitating local delivery of the recombinant retroviruses to specifictarget tissues (Nabel et al, 1990, Science, 249, 1285-1288; Ferry et al,1991, Proc Natl Acad Sci USA, 88, 8377-8381), or retrovirus mediatedgene transfer to target cells ex vivo, followed by reimplantation ofthese cells (Rosenberg et al., 1990, N Engl. J. Meal. 323, 570-578). Analternative strategy would be to alter the host range of the virus(Russell S J (1990) Recombinant viruses expressing lymphokine genes:Their construction and use to modulate growth of transplantable rodenttumours. PhD Thesis London University) which is determined in part bythe binding properties of the proteins displayed at its surface (Marshet al, 1989, Adv Virus Res, 36, 107-151), as discussed above.

The invention will be further described by way of illustration in thefollowing examples and with reference to the accompanying Figures, inwhich:

FIG. 1 illustrates construction of the plasmid pNIPenv.

FIG. 2 illustrates plasmids pNIPenv and pDCNeo, including sequence andtranslation of pNIPenv expression vector showing details of fusionbetween MoMLVenv and B1.8 scFv (SEQ ID NOS: 6-9) LTR=long terminalrepeat; L=33 aminoacid env leader peptide: SV40E=early promoter;enh-=enhancer deletion.

FIG. 3 shows the results of supenatant ELISA for B1.8scFv-MoMLVenvfusion protein binding to NIP.BSA.

FIG. 4 is an electron micrograph (×20,000 magnification) ofNIP.BSA-agglutinated virus from psi2-NIPenv5 supenatant.

FIG. 5 is an IRISA plate (5% Giemsa). Wells were coated as indicated andbound virus was detected by transfer of G418 resistance to NIH3T3 cells.Psi2-NIPenv5-derived virus binds specifically to NIP.BSA-coated wells.

FIG. 6 is an inhibition IRISA plate (5% Giemsa). All wells were coatedwith NIP.BSA. Psi2-NIPenv5-derived virus (1 ml) was preincubated withvarying concentrations of soluble NIP.BSA, as indicated on thephotograph, prior to assay. Virus binding is progressively inhibitedwith increasing concentrations of soluble NIP.BSA.

FIG. 7 is an IRISA plate (5% Giemsa). Wells were coated as indicated andbound virus was detected by transfer of puromycin resistance to NIH3T3cells. Virus expressed by an amphotropic producer cell line infectedwith psi2-NIPenv5-derived virus binds specifically to NIP.BSA-coatedwells.

FIG. 8 illustrates recovery of integrated (proviral) B1.8 scFv codingsequences from GP+envAm12-BabePuro after infection with psi2-NIPenv5virus by PCR amplification.

EXAMPLES Example 1 MATERIALS AND METHODS

Plasmid construction

The BamHI/ClaI env fragment (nt 6537-7674, nt numbering from Shinnick T.M., Lerner R. A., and Sutcliffe J. G. (1981) Nature, 293, 543-548) frompCRIP (gift from O. Danos (Danos O. and Mulligan R. C. (1988) Proc NailAcad Sci USA, 85, 6460-6464)) was cloned into the BamHI/ClaI backbonefragment of pZipNeoSV(X) (gift from R. Mulligan--see Cepko C. L.,Roberts B. E. and Mulligan R. C. (1984) Cell, 37, 1053-1062) to generatean intermediate plasmid penvBam/Cla.

A SfiI/NotI cloning site was introduced beyond the leader peptidesequence between codons corresponding to the 6th and 7th aminoacids(from the N-terminus) in the mature MoMLV env polypeptide. Theoligonucleotides envNotrev (5 '-CTG CAG GAG CTC GAG ATC AAA CGG GCG GCCGCA CCT CAT CAA GTC TAT AAT ATC-3' (Seq. Id. No. 1), complementary toMoMLV env nts 5894-5914 with a 33nt 5' sequence encoding a NotI site and21nt complementary to the 5' tail of envSfifor) and envseq (Nabel E. G.,Plautz G. and Nabel G. J. (1990) Science, 249, 1285-1288) (5'-GCC AGAACG GGG TIT GGC C-3'(Seq. Id. No. 2), complementary to MoMLV env nts6600-6581) were used to amplify a 739 bp fragment from plasmid pCRIP(and encoding downstream of codon 6). A second set of oligonucleotides,envSfifor (5'-TTT GAT CTC GAG CTC CTG CAG GGC CGG CTG GGC CGC ACT GGAGCC GGG CGA AGC AGT-3'(Seq. Id. No. 3), complementary to MoMLV env nts5893-5873 with a 36nt 5' overhang encoding a SfiI site and 21ntcomplementary to the 5' tail of envNotrev) and revMLVpol (5 '-AAT TACATT GTG CAT ACA GAC CC-3'(Seq. Id. No. 4), complementary to MoMLV polnts 5277-5249) was used to prime amplification of a 702bp fragment frompCRIP (and encoding upstream of env codon 7). Amplifications werecarried out using Vent polymerase and reactions were subjected to 15 PCRcycles at 94° C. for 1min, 60° C. for 1min and 72° C. for 1min. The 702and 739 bp gel-purified PCR products were linked through theircomplementary 21nt tails to generate an env gene fragment incorporatinga SfiI/NotI cloning site: the two fragments were mixed and subjected tothree cycles (94° C.-1min, 40° C. -1min, 72° C.-2min) followed by 17further PCR cycles (94° C-1min, 60° C.-1min, 72° C.-2min) after additionof ologonucleotides envseq7 and Bglenvrev (5'-TAA TCA CTA CAG ATC TAGACT GAC ATG GCG CGT-3' (Seq. Id. No. 5), complementary to MoMLV polnucleotides 5766 to 5785 and with the 5' tail incorporating a BglIIrestriction site). The product, a 905 bp fragment, was digested withBglII and BamHI and cloned into the BamHI site of penvBam/Cla (seeabove) giving the plasmid pSfi/Notenv. Correct assembly of this plasmidwas confirmed by restriction analysis and dideoxy sequencing. ASfiI/NotI fragment from pB1.8 scFv, encoding a functional B1.8 scFvantibody was then cloned into the SfiI/NotI cloning site of pSfi/Notenvto generate the plasmid pNIPenv (see FIGS. 1 and 2). Plasmid pDCNeo(FIG. 2, a gift from Dr P Allen, Institute of Cancer Research, FulhamRoad, London) is a retroviral plasmid which carries the bacterialneomycin phosphotransferase gene. It generates packagable RNAtranscripts which are encapsidated into recombinant MoMLV particles andtransfer G418 resistance to infected target cells.

In FIG. 2, the first part of the nucleotide sequence shown is Seq. Id.No. 6, with the corresponding amino acid sequence being Seq. Id. No. 7,and the second part of the nucleotide sequence shown is Seq. Id. No. 8,with the corresponding amino acid sequence being Seq. Id. No. 9.

Cells and recombinant retroviruses

NIH3T3 fibroblasts, the ecotropic retroviral packaging cell line psi2(Mann R., Mulligan R. C. and Baltimore D. (1983) Cell, 33, 153-159) andthe amphotropic retrovirus producer cell line GP+envAm12-BabePuro (agift from R. G. Vile, ICRF, Lincolns Inn Fields, London--derived bytransfection of GP+envAm12 (Markowitz D., Goff S. and Bank A. (1988)Virology, 167, 400-406) cells with the plasmid pBabePuro (Morgenstern J.P. and Land H. (1990) Nucleic Acids Res., 18, 3587-3597) were maintainedin DMEM/10%FBS supplemented with 601μ/ml benzylpenicillin and 100μg/mlstreptomycin at 37° C. in an atmosphere of 5%CO₂. The cells werereplated twice weekly using EDTA without trypsin to disrupt themonolayer.

Plasmids pNIPenv and pDCNeo were transfected (or co-transfected) intopsi2 cells by calcium phosphate precipitation (Graham F. L. and van derEb A. J. (1973) Virology, 52, 456-467). Briefly, 2×10⁵ cells were platedin 90 mm tissue culture plates (Nunc), cultured overnight, washed andfed with 10 mls new medium. 10μl plasmid DNA and 50 μl 2M CaCl₂ (0.2μm-filtered) were diluted in sterile water to a volume of 400 μl. TheCaCl₂ /DNA mi was added dropwise to an equal volume of 0.2 μm-filtered233 HEPES-buffered saline (280 mM NaCl, 10 mM KCl, 1.5 mM Na₂ HPO₄.2H₂O, 12 mM dextrose, 50 mM HEPES, pH adjusted to 7.05 with 0.5N NaOH) andleft to stand for 20 minutes at RT. The transfection solution (800 μl)was added to the cells which were cultured for 16 hrs, washed andre-fed. G418 selection (1 mg/ml) was commenced 24 hrs later andcontinued for approximately 2 weeks.

Transfected colonies expressing surface B1.8 single chain antibody wereidentified by panning with NIP.BSA-coated beads. Briefly, tosylactivated paramagnetic beads (Dynal, Oslo, Norway. Prod. no. 14004) werecoated with NIP.BSA (about 10 NIP-caproate-O-succinimide moleculescoupled to each bovine serum albumin molecule Hawkins R. E., Russell S.J. and Winter G. (1992) J Mol Biol., 226, 889-896), washed extensivelyin PBS and blocked with DMEM/10%FBS. 90 mm tissue culture platescontaining up to 50 G418-resistant psi2 colonies were rocked gently for1 hr at 4° C. followed by 1 hr at room temperature with 2×10⁷ (50 μl)beads in 5 mls DMEM/10%FBS. After 5 washes in PBS, positive colonies(heavily coated with paramagnetic beads) were easily identified and weretransferred individually for further growth and harvest of cellsupernatants.

Recombinant retrovirus titres were determined by transfer of G418 orpuromycin resistance. NIH3T3 cells were infected by overnight exposureto 0.45 μM-filtered viral supernatants in the presence of 5 μg/mlpolybrene and colonies resistant to 1 mg/ml G418 or 1.25 μg/ml puromycinwere counted after 10-14 days. The amphotropic producer cell lineGP+envAm12-BabePuro was infected with ecotropic virus by exposing 10⁵cells overnight (twice) to 10 mls of the appropriate producer cellsupernatant in the presence of 5 μg/ml polybrene.

ELISA for B1.8scFv-MoMLVenv fusion protein

To detect the B1.8-env fusion protein in supernatant of pNIPenvtransfected clones, 96-well microtiter plates (Falcon) were coatedovernight at RT with 20 μg/ml NIP.BSA or BSA alone, blocked for 2 hrs at37° C. in DMEM/10%FCS and washed ×6 in PBS. Culture supernatants,cleared of cell debris by centrifugation at 5000 RPM for 15 min, wereadded in triplicate to coated wells and incubated for 2 hrs at RT. Wellswere washed (PBS×6). The second layer was a goat polyclonalanti-Rauscher MLV env antiserum (Microbiological Associates, Inc.Bethesda), diluted 1/500 in DMEM/10%FCS, and incubated at RT for 1 hr.After 6 washes in PBS, the third layer HRP-conjugated rabbit anti-goatantibody (Sigma) was added, the plates incubated for a further hour atRT, washed ×6 in PBS and the reaction developed with ABTS(2'2'-azinobis(3-ethylbenzthiazolone)sulphonic acid). Absorbancereadings were measured at 405 nm after 20 minutes.

Infectious retrovirus immunosorbent assay (IRISA)

Individual wells in 6-well tissue culture plates (Coming, N.Y.) werecoated overnight at 4° C. with 100 μg/ml NIP.BSA or Ox.BSA (about 14molecules 2-phenyl-5-oxazolone coupled to each bovine serum albuminmolecule, and was a gift from C.Rada), washed 3×PBS, blocked for 2 hrsat 37° C. with DMEM/10%FBS and washed 3×PBS. Virus-containingsupernatant (0.45 μM-filtered) was added (2 hrs at 37° C.), wells werewashed 6×PBS and 10⁵ NIH3T3 cells were added to each well in 5 mlsDMEM/10%FBS containing 5 μg/ml polybrene. After 24 hrs incubation, G418or puromycin was added, either before or after replating. 10-14 dayslater, viable colonies were stained with 50% methanol/5% Giemsa andcounted. For inhibition IRISA, virus-containing supernatants werepre-incubated (30 mins at room temperature) with varying concentrationsof NIP.BSA.

EM analysis of virus agglutination 0.45 μM-filtered virus-containingsupernatants were incubated overnight at 4° C. with varyingconcentrations of NIP.BSA. Virions were pelleted by centrifugation at40,000 rpm for 1 hr, resuspended in 100 μl 2% phosphotungstic acid anddropped onto Formvar-coated grids which had previously been coated witha thin layer of carbon. Transmission electron micrographs were takenwith a Joel JEM100CX microscope at magnifications ranging from 10,000 to50,000.

RESULTS

Design of pNIPenv vector

Plasmid pNIPenv (FIG. 2) encodes a chimaeric fusion protein consistingof the ecotropic MoMLV envelope polypeptide Pr80env with a single chainFv (scFv) (Huston J. S., Levinson D., Mudgett H. M., Tai M. S., NovotnyJ., Margolies M. N. et al. (1988) Proc Nail Acad Sci USA, 85, 5879-5883;Bird R. E., Hardman K. D., Jacobsen J. W., Johnson S., Kaufman B. M.,Lee S. M. et al. (1988) Science, 242, 423-426) fragment directed againstthe hapten 4-hydroxy-5-iodo-3-nitrophenacetyl caproate (NIP) Hawkins etal. 1992, loc cit) inserted 6 aminoacids from the N-terminus of Pr80env.The scFv fragment is flanked by SfiI and NotI sites as in the vectorpHEN1 (Hoogenboom H. R., Griffiths A. D., Johnson K. S., Chiswell D. J.,Hudson P. and Winter G. (1991) Nucleic Acids Res., 9, 4133-4137 ), tofacilitate the cloning of scFv fragments selected from phage displaylibraries (Marks J. D., Hoogenboom H. R., Bonnert T. P., McCafferty J.,Griffiths A. D. and Winter G. (1991) J Mol Biol, 222, 581-597). The 33aminoacid MoMLV env leader sequence is retained, without disruption ofthe leader cleavage site. The N- and C-termini of the B1.8scFv areconnected to adjacent env sequences through short linker sequences (FIG.2). Expression is driven from promoter/enhancer sequences in the 5'MoMLV long terminal repeat (LTR) and a polyadenylation sequence isprovided by the 3' MoMLV LTR.

Display of antibody fragments on surface of mammalian cells

Plasmid pNIPenv was co-transfected with the retroviral plasmid pDCNeo(which generates a packagable RNA transcript encoding neomycinphosphotransferase and confers G418-resistance, FIG. 2) into theecotropic retroviral packaging cell line psi2. Control cells weretransfected with pDCNeo alone. G418-resistant psi2 transfectantsdisplaying the B1.8 scFv-MoMLVenv fusion protein at their surface wereidentified by panning with NIP.BSA-coated paramagnetic beads. The cellsisolated by panning were heavily coated with the beads, indicating thata functional antibody fragment was displayed on the surface of thetransfected cells.

Display of antibody fragments on surface of retrovirus

The retroviruses expressed from the selected clones were titred bytransfer of G418 resistance to NIH3T3, and a range of titres noted, forexample clones psi2-NIPenv1 (titre 0 G418 t.u./ml) and psi2-NIPenv5(titre 10³ G418 t.u./ml). Cell supernatants were then tested by ELISAfor presence of the B1.8scFv-env fusion protein (FIG. 3). Using anti-envantiserum as the second layer, specific NIP.BSA-binding activity wasdetected in supernatants from pooled pNIPenv psi2 clones (titre 10³ G418t.u./ml) and from clone psi2-NIPenv5, but not from clone psi2-NIPenvl orpooled psi2-DCNeo clones (titre 10³ G418 t.u./ml). This suggested thatthe functional antibody fragment could be incorporated into virionparticles and displayed at their surface.

As a further demonstration, 0.45 μM-filtered culture supernatantpsi2-NIPenv5-virus was incubated with varying concentrations (0, 0.1,1.0 and 10.0 μg/ml) of NIP.BSA and the resuspended viral pellet examinedby electron microscopy for virus agglutination. At 10 μg/ml NIP.BSA,numerous large aggregates of MoMLV particles with typical morphology(Dalton A. J., Haguenau F. and Moloney J. B. (1964) J Natl Cancer Inst.,33, 255-275) were observed (FIG. 4). Individual virions were closelyapposed with a relatively uniform interparticle distance (6-20 nm),indicating crosslinking by NIP.BSA. Similar aggregation was not observedin the absence of NIP.BSA, nor with control psi2-DCNeo supernatant.

Retrovirus particles displaying antibody fragments can package markergenes

As proof that the functional antibody fragment was displayed oninfectious retroviral particles, the novel IRISA assay was developed(Materials and Methods). Filtered recombinant psi2-NIPenv5 virus wasbound to NIP.BSA-coated plates, washed and NIH3T3 cells added to eachwell. The bound virus gave rise to G418 resistant colonies (FIG. 5 andTable 1), and the binding of the virus could be competively inhibited bysoluble NIP.BSA (FIG. 6). The virus did not bind to phOx-BSA-coated oruncoated tissue culture wells, nor did psi2-DCNeo virus bind to NIP.BSA.This indicates that the virus particles bind specifically to hapten, areinfectious and can package a marker gene for transfer into mousefibroblasts.

Retroviral particles displaying antibody fragments can package theantibody V-genes

The amphotropic producer cell line GP+envAm12-BabePuro was infected withrecombinant psi2-NIPenv5 virus and transfer of the hybrid B1.8scFv-MoMLVenv fusion gene was confirmed by PCR analysis of high molecular weightDNA extracted from these cells. Results are shown in FIG. 8. Highmolecular weight DNA was extracted from the infected cells and amplifiedusing PCR primers VH1BACK (Orlandi et al., 1989 Proc Nail Acad Sci USA86 p3833-3837) and envseq5 (5¹ - GTA AGG TCA GGC CAC GAG GT-3¹ (Seq. Id.No. 10)), reverse complement of MoMLV env nucleotides 5981-5600.Amplification was carried out using Promega Taq polymerase and reactionswere subjected to 30 cycles at 94° C. for 1 min, 55° C. for 1 min and72° C. for 1 min. In FIG. 8, Lane 1=PhiX174 Hae III digest (fragmentsizes 1353, 1078, 972, 603, 310, 281,271, 234, 194, 118, 72). Lane2=GP+envAm12-BabePuro infected with 10 mls psi2-NIPenv5 virus. Lane3=Uninfected GP+envAm12-BabePuro. As a demonstration of functionalexpression of the transferred env fusion gene, the virus particlesexpressed by these cells bound more efficiently to NIP.BSA-coated platesthan to phOx.BSA-coated plates as indicated by subsequent transfer ofpuromycin resistance to NIH3T3 cells (FIG. 7). In contrast,GP+envAm12-BabePuro cells infected with Psi2-DCNeo virus gave no signalwhen analysed by PCR for the env fusion gene and expressed virus whichdid not bind specifically to NIP.BSA. This indicates thatpsi2-NIPenv5-derived virus particles encapsidate and transfer afunctional gene encoding the functional B1.8scFv antibody fragmentdisplayed on their surface.

DISCUSSION

We have shown that a functional antibody fragment can be displayed onthe surface of a retroviral particle fused to its envelope protein, andthat this confers novel binding specificity on the particle. Pr85env,the initial translation product of the env gene forms oligomers,undergoes glycosylation and is proteolytically cleaved during itstransport through the endoplasmic reticulum and Golgi apparatus to thecell surface where it appears as a small transmembrane C-terminal domainp15(E)TM, linked noncovalently or by a disulphide bridge to a largerextracellular domain gp70SU (Weiss R., N. Teich, H. Varmus and J. Coffin(eds). (1985) RNA turnout viruses, volume 2. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Here we fused the scFv fragmentclose to the N-terminus of gp70SU and we envisage that it is folded anddisplayed as a separate domain. This choice of fusion site may beimportant, as an attempt to replace the N-terminal domain of gp70SU witha functional IL2 polypeptide did not succeed (Russell S. J. (1990)Recombinant viruses expressing lymphokine genes: Their construction anduse to modulate growth of transplantable rodent turnouts. PhD Thesis.London University). Presumably it will prove possible to incorporateantibody fragments with different binding specificities, and indeed therestriction sites of pNIPenv were designed to facilitate the cloning ofother scFv cassettes from filamentous phage vectors (Marks et at., 1991,loc cit). It may also be possible to display other functional nonviralpolypeptides (growth factors, cytokines or T cell receptors for example)or short peptide sequences with a variety of receptor bindingactivities.

We showed that the virus particles that display antibody fragments couldalso encapsidate the genes of a marker (neo), and were infectious asshown by the transfer of G418 antibiotic resistance to murine cells fromviral particles immobilised on NIP-BSA coated plates. The infectivity ofthese particles was expected since they incorporate both theantibody-envelope fusion protein and unmodified envelope protein whichis also expressed abundantly in the retroviral packaging cell line. Inprinciple such particles could be used for targeted delivery of agenetic marker to cells (see below). It is not known whether retroviruscan be assembled in which all the subunits of the viral envelope proteinare fused to antibody, and if so whether the virus would infect cells.

We also demonstrated that virus particles displaying antibody fragmentscan encapsidate the genes (pNIPenv) encoding the antibody fragments ontheir surface and hence have potential as replicable display packages,as with phage antibodies (McCafferty et al., 1990, loc cit). (Thesevirions could also have been selected directly by including a selectablemarker gene on the pNIPenv plasmid). We suggest that it might bepossible to evolve new viral tropisms (see below) using repertoires ofantibody fragments or peptide sequences displayed on the virus, withcycles of infection and selection.

Retroviral particles displaying antibodies against cell surface antigensshould bind preferentially to target cells expressing those antigens,and this may facilitate their infection. For some antigens, the bindingof retrovirus-associated antibody fragments to cell surfaces is followedby membrane fusion between virus and target cell: streptavidin-linkedbiotinylated monoclonal antibodies have been used to link ecotropicretroviruses to the surface of nonpermissive human cells with subsequenttransfer of the viral genome into the target cells (Roux P., Jeanteur P.and Piechaczyk M. (1989) Proc Natl Acad Sci USA, 86, 9079-9083). This"molecular bridging" approach was successful (but inefficient) whenviral particles were coupled to human MHC class I and class II antigens,but not to the human transferrin receptor (Gould B., Legrain P. andButtin G. (1988) Virology, 163, 251-254), and suggests that only alimited number of cell surface antigens can function as surrogatereceptors for MoMLV particles. In an attempt to identify suitablesurrogate receptors, we are currently generating ecotropic retroviralparticles displaying antibody fragments against a number of targetantigens present on human cells. NIP-derivatised human cells were testedas a model for targeted gene delivery, but became permissive for bothmodified (displaying an anti-NIP antibody) and unmodified ecotropicviral particles.

Retroviruses displaying antibody fragments might also be used to retainthe retrovirus in the vicinity of a tumour, and thereby reduce thesystemic spread of recombinant retroviruses. For example, it has beenproposed to deliver genes encoding prodrug-activating enzymes to tumoursby injection of retroviral vector-producer cells (Culver K. W., Ram Z.,Wallbridge S., Ishii H., Oldfield E. H. and Blaise R. M. (1992) Science,256, 1550-1552; Stone R (1992) Science, 256, 1513), and then toadminister the appropriate prodrug to kill gene-transduced tumour cellsand their untransduced neighbours. As a safety measure, the retroviralparticles could be engineered to display antibody fragments directedagainst an antigen on the tumour cells to enhance their retention withinthe tumour deposit.

Example 2

Host Range Modification Example

A431 is a human cell line expressing abundant EGF receptors and themonoclonal antibody 425 binds specifically to human EGF receptors. ApHen-1 derived plasmid encoding the 425 antibody as a functional, EGFreceptor-binding single chain Fv was obtained from Detlef Gussow (MRCCollaborative Centre). The 425 scFv gene was cloned as a SfiI-NotIfragment into pNIPenv in place of the B1.8 scFv. The resulting plasmid(pEGFRenv) was contransfected with plasmid pBabePuro into ecotropicretrovirus packaging cells. Colonies resistant to 1.25 μ per mlpuromycin were either pooled or expanded individually and supenatantderived from these cells was passed through a 0.45 μM filter and testedfor its ability to transfer puromycin resistance to human A431 cells.A431 cells were infected by overnight exposure to virus-containingsupenatant in the presence of polybrene (8 μg/ml) and were selected for21 days in 0.6 μg/ml puromycin. Supernatants derived from ecotropicproducer cells transfected with pEGFRenv repeatedly gave low efficiencytransfer of puromycin resistance to A431 cells (approximately onepuromycin resistant colony per ml of supernatant). Control virus derivedfrom the same ecotropic packaging cells transfected with pBabePuro alonedid not transfer puromycin resistance to A431 cells. We conclude fromthese results that retrovirus particles displaying an anti-human EGFreceptor antibody fragment in fusion with the viral envelope proteinhave an extension to their host range and are able to infect otherwisenonpermissive cells which express the human EGF receptor.

Example 3

Possible examples of novel selection strategies

1. This example teaches how to select for a nonviral polypeptide which,when displayed on a recombinant virus, increases the efficiency withwhich the recombinant virus delivers the viral nucleic acid to a humantarget cell through a target cell-selective entry pathway which leads tothe expression of the viral nucleic acid.

In this example, the experimental conditions are set such that theviruses contained in the library display no viral surface proteinscapable of efficient binding to the target cells. The specificinteraction of viruses in the library with the surface of the targetcells and the subsequent delivery of the viral nucleic acid is thereforepossible only if mediated by the virus-displayed nonviral polypeptide.

Single-chain antibodies are pre-selected from a large (10¹⁰ diversity)random combinatorial phage antibody library for binding to the surfaceof the cell line K422 (Dyer et al, 1990 Blood 75 p709-714) and aresubcloned as SfiI-Not I fragments into into the vector pNIPenv.

The vector plasmid library is introduced by lipofection into the psi-2packaging cell line (Mann et al, 1983 Cell 33 p153-159) and library ofrecombinant retroviruses is harvested in the form of cell culturesupernatant after 72 hrs. The estimated library diversity is 10⁴ membersand the estimated virus titre is 10⁵ infectious particles per ml ofculture supenatant. The unmodified spike glycoproteins displayed by theviruses and the viral moieties of the modified spike glycoproteins(which are fused to single chain antibody domains) are both mouseecotropic MoMLV envelope proteins which do not bind to human cellsunless they have been genetically modified to express the gene encodingthe ecotropic retrovirus receptor (Albritton et al, 1989 Cell 57p659-666; Albritton et al, 1993 J Virol 67 p2091-2096).

The retrovirus display library is pre-adsorbed on the human myeloma cellline Karpas 620 (Nacheva et al, 1990 Brit J Haem 74 p70-76) which is ofB cell origin and derives from a patient with plasma cell leukaemia. 10⁷Karpas 620 cells are added to 10 mls of 0.2 μm-filtered viruslibrary-containing supernatant (RPMI/10% FCS plus antibiotics) with 8 μgper ml polybrene 4° C. for 4 hours for virus preadsorption.

The pre-adsorbed virus library is then separated from the Karpas 620cells by centrifugation and mixed with 10⁶ K422 cells (Dyer et al, 1990Blood 3 p709-714) at 4° C. for 2 hours to allow virus binding, moving to37° C./5% CO₂ for virus entry. K422 is a human B-cell line derived froma patient with follicular B-cell non-Hodgkin's lymphoma.

The viruses display only murine ecotropic virus spike glycoproteins(which do not bind human target cells) and modified murine ecotropicvirus spike glycoproteins fused to single-chain Fvs pre-selected forbinding to the B-cell line K422 which has the immunophenotype of afollicle centre cell. They will not deliver their encapsidated nucleicacid to the human K422 target cells unless they display a single chainFv which can interact with a suitable receptor site on these cells whichis not present on the plasma cell line Karpas 620.

The retrovirus display library is pre-adsorbed on a human plasma cellline, also of B cell lineage but more differentiated than the folliclecentre cell. Viruses which remain after preadsorption are likely to becapable of infecting the K422 cells only through binding to the K422idiotypic surface immunoglobulin (a unique tumour-specific antigen) orto differentiation antigen present on the K422 cells, but lacking on theplasma cells. Such differentiation antigen are likely to be specific tothe B-cell lineage.

The infected K422 cells are refer and expanded in tissue culture for 7days, stained with mouse monoclonal antibodies or goat polyclonalantisera against the MoMLV envelope spike glycoprotein, withsecond-layer fluorescent anti-mouse Fc or anti-goat Fc antibodies andpositively staining cells are isolated by FACS sorting.

The viral sequences coding for a displayed single chain Fv which bindsto the K422 cells, leading to viral delivery of the nucleic acid arerecovered by PCR amplification from chromosomal DNA extracted from the(selected or unselected) cells, using the PCR primers BglenvRev andenvseq5. The PCR product is digested with Sfi I and Not I, purified onan agarose gel and recloned in the SfiI-Not I--digested pNIPenv backboneto generate a secondary vector library which is used to generate asecondary retrovirus display library by transfection into the psi-2packaging cell line. The secondary retrovirus display library issubjected to a second round of the same selection procedure and thenucleic acid sequences coding for the selected single chain Fvs arerecovered as previously.

At this stage the PCR product may be diversified according to a varietyof previously demonstrated strategies, recloned into pNIPenv and used togenerate further retrovirus display libraries for use in further roundsof selection. Thus the invention provides for methods to evolve apolypeptide which is displayed on its surface using repeated rounds ofdiversification and selection.

At any stage after the first round of selection, individual selectedsingle chain antibody sequences can be isolated, subjected to sequenceanalysis, cloned into expression vectors and used to produce solubleantibody fragments or cloned into pNIPenv and used to produce a purepopulation of retroviruses displaying a single nonviral polypeptide. Thepurified antibody fragments and recombinant viruses can then be used todetermine the identity and tissue distribution of the target antigen towhich the selected single chain Fv binds.

The choice of nontarget cells (or cloned antigens) for the preadsorptionstep can be varied to suit the precise goals of the selection procedure.Thus, pre-adsorption on a B-cell line derived from another patient withfollicular NHL, an early B-lineage cell line, the cloned K422 idiotypicimmunoglobulin, a non-B cell line or a combination of different celllines or cell lines and antigen could be used to skew the selection.

The invention therefore provides for methods to select single chainantibodies or other peptides, polypeptides or glycopolypeptides whichmay be used to target virus-mediated gene delivery using nonreplicatingor replicating recombinant viruses to tumour cells, stem cells, or moredifferentiated cells from any human tissue. Targeting efficiency couldbe further increased by incorporating tissue-specific ortumour-selective promoters, enhancers, silencers or locus-controlsequences into such recombinant viruses. The selected peptides,polypeptides or glycopolypeptides could also be used to target genedelivery by nonviral gene delivery vehicles, for example by conjugationto plasmid DNA or by incorporation into cationic liposomes. They mayalso be useful as components of diagnostic kits or as tools to clonecellular receptors or as components of protein-based therapeuticreagents for targeted therapy of a variety of diseases including cancer,autoimmunity and AIDS.

2. The experimental conditions may also be set such that the virusesfrom which the library is derived are infectious for the target cellsbut increased efficiency of infection desired. Viruses in the displaylibrary are selected according to the efficiency with which they deliverthe nucleic acid to the target cells. Murine amphotropic retroviralvectors, for example commonly give 10-fold reduced titre on humancompared to murine cells and it is desirable to select for displayedpolypeptides which overcome this deficiency. In this case, negativeselection on human nontarget cells is not possible. Multiple rounds ofselection and amplification are therefore required to enrich the librarywith members which infect the target cells most efficiently. Forexample, using a murine amphotropic retroviral packaging cell and thepNIPenv vector, it is possible to generate a-library of virusesdisplaying single chain antibodies pre-selected (on phage) for bindingto a T-cell line such as Jurkat and then to complete multiple rounds ofselection for members of the library which infect the line mostefficiently. To avoid the labour of repeatedly recloning isolated PCRfragments and retransfecting the packaging cells, it is possible toemploy Jurkat cell targets stably transfected with plasmids encodingretroviral gag, pol and, xenotropic or amphotropic env proteins suchthat they encapsidate the delivered retroviral nucleic acid into newretroviruses for subsequent rounds of selection (alternating thexenotropic and amphotropic rescue). Diversification of the librarybetween rounds of selection provides for directed evolution of thedisplayed polypeptides.

3. The selection conditions may also be set such that the viruses fromwhich the library is derived are capable of binding to the target cellsbut the target cells are not dividing and can be stimulated to divide byexposure to a very low concentration of a (known or unknown) growthfactor. Where the growth factor is known, the initial library of virusesmay display a diverse population of artificially generated geneticvariants of the growth factor. Where the growth factor is poorlydefined, the initial library of displayed nonviral polypeptides may bederived from a cDNA library obtained from a cell line known to producethe factor. Following exposure to a library of retroviruses the cellsare selected for expression of a marker gene delivered by the retroviralnucleic acid, a phenotype which indicates that reverse transcription andintegration of the retroviral nucleic acid has occurred and that thetarget cell has therefore undergone cell division. Alternatively, theintegrated proviral sequences can be recovered directly by PCRamplification from the high molecular weight chromosomal DNA of thetarget cells. The method can be used to isolate a nonviral polypeptidewhich triggers division of target cell in the context of viral particle.Again, diversification between rounds of selection provides for directedevolution of selected members of the original library.

4. In order to evolve a new virus tropism, the experimental conditionsare set such that the viruses contained in the library arereplication-competent and display no viral surface proteins capable ofefficient binding to the target cells. The specific interaction ofviruses in the library with the surface of the target cells and thesubsequent delivery of the viral nucleic acid is therefore possible onlyif mediated by the virus-displayed nonviral polypeptide. Using afull-length infectious molecular clone of MoMLV, for example whichincludes Sfi I and Not I restriction sites as in pNIPenv, a variegatedlibrary of constructs encoding infectious (mouse ecotropic) virionswhich display different nonviral peptides/polypeptides/glycopolypeptidesis generated. The library is applied to the human target cells withnegative selection (pre-adsorption) steps on carefully chosen humannontarget cells and virus progeny are harvested after 48 hours andsubjected to repeated rounds of the same selection procedure.

                  TABLE 1                                                         ______________________________________                                        Pooled IRISA data for psi2-NIPenv5- and psi2-DCNeo-derived virus              binding to NIP.BSA or OX.BSA. Numbers represent Giemsa-stained                G418 resistant colony counts (halved for assays which were replated           after 24 hrs).                                                                       Input      NIP.BSA  Ox.BSA                                             ______________________________________                                        NIPenv5                                                                                1950          14      2                                                       1950          20      1                                                        900          31      0                                                       1560         100       12*                                                    1560          80       11*                                                    4650         125      1                                                       5950         150      2                                              Totals   18520        520       29                                            DCNeo                                                                                  1100          9        7*                                                      625          0       0                                                        625          0       0                                                       1375          2       4                                              Totals   3725          11       11                                            ______________________________________                                         *polybrene added to initial virus binding reaction                       

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CTGCAGGAGCTCGAGATCAAACGGGCGGCCGCACCTCATCAAGTCTATAATATC54                      (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      GCCAGAACGGGGTTTGGCC19                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      TTTGATCTCGAGCTCCTGCAGGGCCGGCTGGGCCGCACTGGAGCCGGGCGAAGCAGT57                   (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      AATTACATTGTGCATACAGACCC23                                                     (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      TAATCACTACAGATCTAGACTGACATGGCGCGT33                                           (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      ACTGCTTCGCCCGGCTCCAGTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCAG57                   (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ThrAlaSerProGlySerSerAlaAlaGlnProAlaMetAla                                    1510                                                                          GlnValGlnLeuGln                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      GTCCTCGAGGAGGCGGCCGCACCTCATCAAGTCTAT36                                        (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      ValLeuGluGluAlaAlaAlaProHisGlnValTyr                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iii) ANTI-SENSE: NO                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     GTAAGGTCAGGCCACCAGGT20                                                        __________________________________________________________________________

We claim:
 1. A recombinant retroviral particle that infects eukaryoticcells, comprising (a) a fusion protein comprising a non viralpolypeptide fused at the amino-terminus or within seven amino acidresidues of the amino terminus of a substantially intact viralglycoprotein said non viral polypeptide being, displayed on the externalsurface of the particle; and (b) a nucleic acid encoding said fusionprotein, the nucleic acid further comprising a packaging signalsequence.
 2. The particle according to claim 1, wherein the viralglycoprotein comprises a spike glycoprotein.
 3. The particle accordingto claim 1, further comprising one or more viral coat proteins.
 4. Theparticle according to claim 1 that infects human cells.
 5. A recombinantretroviral particle that infects eukaryotic cells, comprising a nonviral polypeptide fused at the amino-terminus or within seven amino acidresidues of the amino terminus of substantially intact viralglycoprotein and displayed on the external surface of the particle. 6.The particle according to claim 5, wherein the viral glycoproteincomprises a spike glycoprotein.
 7. The particle according to claim 5,further comprising one or more viral coat proteins.
 8. The particleaccording to claim 5, that infects human cells.
 9. The particleaccording to claim 5, wherein the non viral polypeptide binds to a cellsurface molecule of target eukaryotic cells.
 10. The particle accordingto claim 5, wherein the non viral polypeptide comprises an antibody orantibody fragment.
 11. A library of retroviral display packagescomprising a plurality of said recombinant retroviral particle of claim5.
 12. The library according to claim 11, wherein the displayed nonviral polypeptides are glycoproteins.
 13. A method of identifying anucleic acid of a retroviral display package in the library ofretroviral display packages of claim 11 which is delivered into aeukaryotic cell by the package, comprising contacting packages from thelibrary with target cells, and isolating delivered nucleic acid from thetarget cells, such that said nucleic acid is identified.
 14. A method ofidentifying a nucleic acid of a retroviral display package in thelibrary of retroviral display packages of claim 11 which is deliveredinto a eukaryotic cell by the package, comprising contacting packagesfrom the library with target cells, and detecting proviral DNA in thetarget cells, such that said nucleic acid is identified.
 15. A method ofidentifying a nucleic acid of a retroviral display package in thelibrary of retroviral display packages of claim 11 which is deliveredinto a eukaryotic cell by the package, comprising contacting packagesfrom the library with target cells, and detecting integrated proviralDNA in the target cells, such that said nucleic acid is identified. 16.A method of identifying a nucleic acid of a retroviral display packagein the library of retroviral display packages of claim 11 which isdelivered into a eukaryotic cell by the package, comprising contactingpackages from the library with target cells, isolating target cells thatexpress a delivered nucleic acid, and amplifying proviral DNA containedin said isolated target cells, such that said nucleic acid isidentified.
 17. A method of identifying a nucleic acid of a retroviraldisplay package in the library of retroviral display packages of claim11 which is delivered into a eukaryotic cell by the package, comprisingcontacting packages from the library with quiescent target cells, anddetecting target cells undergoing mitosis, such that said nucleic acidis identified.
 18. The method of any one of claims 13-17 wherein saidisolating, said detecting or said amplifying steps comprise PCRamplification of said delivered nucleic acid or said proviral DNA. 19.The method of any one of claims 13-17 wherein said delivered nucleicacid or said proviral DNA encodes a detectable product, and saidisolating, said detecting or said amplifying steps comprises detectingsaid product.
 20. A DNA construct suitable for generation of a libraryof retroviral display packages, comprising a nucleotide sequenceencoding a substantially intact viral glycoprotein containing a site forinsertion at the amino terminus or within seven amino acids of the aminoterminus of said substantially intact viral glycoprotein of a sequencefrom a library of sequences encoding non viral polypeptides that permitsin-frame fusion of the non viral polypeptide to said viral glycoproteinto form a fusion protein that is displayed on the external surface of aviral particle; and a suitable packaging signal sequence.
 21. Theconstruct according to claim 20, wherein the site for insertioncomprises a cloning site.
 22. The construct according to claim 20,wherein the glycoprotein is a substantially intact viral glycoprotein.23. The construct according to claim 20, comprising in the 5' to 3'direction: a 5' LTR, a packaging signal sequence, a leader sequence, anSfi I recognition site, a NotI recognition site, a sequence encoding asubstantially intact retroviral envelope glycoprotein, and a 3' LTR. 24.The construct of claim 21, said site for insertion including two uniquenon-complementary restriction sites.